Annotation and functional prediction of RNA helicases in Ustilago maydis

1 Annotation and functional prediction of RNA helicases in Ustilago maydis Amanda M. Seto1, Barry J. Saville2 1 Environmental and Life Sciences Graduate Program, Trent University, Peterborough, ON, K9L 0G2, Canada 2 Department of Forensic Science, Trent University, Peterborough, ON, K9L 0G2, Canada Corresponding author: Barry J. Saville ([email protected]) Copyright: © Amanda M. Seto & Barry J. Saville. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Review Article Abstract RNA helicases are conserved enzymes found in both prokaryotes and eukaryotes. They function in all aspects of RNA metabolism and are known to influence various cellular and metabolic processes. In addition, they have been implicated in certain cancers and diseases. Studies on RNA helicases in fungi indicate their conserved roles in RNA metabolism and suggest that their dysregulation can affect fungal growth. However, the roles of RNA helicases in fungal plant pathogenesis remain underexplored, despite increasing knowledge of how RNA helicases modulate gene expression and disease progression. We used the basidiomycete plant pathogen Ustilago maydis as a model to identify 46 RNA helicases. We review the roles of RNA helicases in RNA metabolism, cellular growth and homeostasis, and metabolism. We then utilized available U. maydis transcriptome data and current research to hypothesize potential functions of RNA helicases in fungal plant pathology. These roles include influencing cell growth, modulating stress response, contributing to virulence and disease progression, and regulating fungal spore dormancy and germination. Understanding the roles of RNA helicases in gene regulation may aid in developing strategies to mitigate disease spread in fungal plant pathogens. Key words: Control of pathogenic development, genome annotation, RNA helicases, smut fungi, Ustilago maydis Introduction Helicases are conserved enzymes that utilize ATP to bind and remodel nucleic acids. These proteins are found in both prokaryotes and eukaryotes, and many exhibit conserved functions across species. DNA helicases participate in several cellular processes, including DNA replication and repair. RNA helicases are involved in all aspects of RNA metabolism, such as transcription, splicing, ribosome biogenesis, and translation (Singleton et al. 2007). A considerable amount of research supports the roles of RNA helicases in cellular metabolism, growth, and viability. An increased understanding of how defective helicases affect cellular processes has also led to the identification of their roles in certain cancers and diseases (Fairman-Williams et al. 2010; Cai et al. 2017). In fungi, functional analysis of RNA helicases has been extensively conducted in the budding yeast Saccharomyces cerevisiae. Investigating their roles in yeast RNA metabolism Academic editor: Michael Bradshaw Received: 1 March 2025 Accepted: 24 June 2025 Published: 2 October 2025 Citation: Seto AM, Saville BJ (2025) Annotation and functional prediction of RNA helicases in Ustilago maydis. IMA Fungus 16: e151785. https://doi. org/10.3897/imafungus.16.151785 IMA Fungus 16: e151785 (2025) DOI: 10.3897/imafungus.16.151785 2 IMA Fungus 16: e151785 (2025), DOI: 10.3897/imafungus.16.151785 Amanda M. Seto & Barry J. Saville: U. maydis RNA helicases has led to the identification of RNA helicases involved in cellular growth, metabolism, and stress response. Although studies on pathogenic fungi are limited, they have revealed roles for RNA helicases in virulence (Panepinto et al. 2005; Bleichert and Baserga 2007; Delaney et al. 2013; Bohnsack et al. 2023). In all of these roles, the helicases act to unwind double-stranded RNA (dsRNA). The separation of RNA–RNA and RNA–DNA duplexes by RNA helicases may also involve displacing proteins from RNA, acting as RNA clamps, or annealing RNA strands. Some RNA helicases are essential, and defects in these proteins can affect cell viability (Bleichert and Baserga 2007; Jankowsky 2011; Linder and Jankowsky 2011), indicating potential roles in cellular growth and homeostasis. In fungi, certain RNA helicases have been shown to modulate the expression of subsets of genes, which may influence stress responses or virulence. For example, in Cryptococcus neoformans, the RNA helicase Vad1 regulates several virulence-associated genes (Panepinto et al. 2005). Characterization of ski2 deletion mutants in C. neoformans revealed decreased virulence, increased resistance to azoles, and sensitivity to high temperatures and osmotic stress (Li et al. 2022). In Neurospora crassa, the RNA helicase FRH forms a complex with FRQ and CK1a to regulate the circadian clock (Cheng et al. 2005; Lauinger et al. 2014). In S. cerevisiae, the nutrient stress response is modulated by the RNA helicase Dbp2 (Beck et al. 2014), which interacts with SKS1, a gene encoding a protein responsible for adaptation to low-glucose conditions (Paul et al. 2025). Despite current knowledge of RNA helicases in fungi, their functions in phytopathogenic fungi remain largely unexplored. Their roles in fungal growth, virulence, pathogenesis, and spore dormancy have not been extensively studied, and experimental investigations in smuts and rusts of the Basidiomycota are limited. In this review, we utilize the smut fungus Ustilago maydis to identify potential RNA helicases of interest and hypothesize their roles in the fungal life cycle. U. maydis is a member of the Basidiomycota and belongs to the Ustilaginales, which includes more than 1,500 species. These species are important plant pathogens that infect members of the plant family Poaceae, which includes many cereal crops. Ustilago maydis infects the aerial parts of maize, causing common smut. These infections result in significant losses in food production, costing millions of dollars annually (Martínez-Espinoza et al. 2002; Brefort et al. 2009). Although its agricultural impact is not as substantial as that of rust fungi, U. maydis is considered one of the top ten fungal pathogens of scientific importance and serves as a model organism for studying plant–pathogen interactions (Dean et al. 2012). This review highlights the functions of RNA helicases in RNA metabolism, identifies RNA helicases in U. maydis, and discusses those that may affect fungal growth, metabolism, stress response, pathogenesis, and teliospore dormancy and germination. Characteristics of SF1 and SF2 RNA helicase superfamilies Currently, six helicase superfamilies containing both DNA and RNA helicases have been identified (Fig. 1A). The largest among these are superfamilies 1 (SF1) and 2 (SF2), with the majority of eukaryotic RNA helicases classified under SF2. The remaining superfamilies (SF3–SF6) typically include enzymes involved in DNA metabolism (Singleton et al. 2007; Fairman-Williams 3 IMA Fungus 16: e151785 (2025), DOI: 10.3897/imafungus.16.151785 Amanda M. Seto & Barry J. Saville: U. maydis RNA helicases et al. 2010). Superfamily 3 (SF3) helicases are found in DNA and RNA viruses (Hickman and Dyda 2005), while superfamily 4 (SF4) helicases occur in bacteria and bacteriophages (O’Donnell and Li 2018). Superfamily 5 (SF5) includes the bacterial RNA helicase Rho, which is responsible for transcription termination (Selvaratnam et al. 2025). Superfamily 6 (SF6), also known as the AAA+ superfamily, comprises helicases involved in DNA metabolism (Ilves et al. 2010). The classification of helicases was first proposed based on sequence and structural motifs by Gorbalenya and Koonin (1993) and was later refined through further structural and functional analyses (Singleton et al. 2007). These sequence motifs make up the helicase core, which contains two RecA-like domains. The functions of these motifs include ATP binding and hydrolysis, RNA binding, interdomain interactions, and coordination between ATP and RNA binding (Bleichert and Baserga 2007; Fairman-Williams et al. 2010). In RNA helicases, the helicase core is characterized by approximately 14 sequence motifs, as illustrated in Fig. 1B. Some motifs (e.g., I, II, and VI) are conserved across all RNA helicases, whereas others are present only in specific subsets (Fairman-Williams et al. 2010; Sloan and Bohnsack 2018). This review focuses on fungal RNA helicases belonging to the SF1 and SF2 superfamilies. Sequence and structural analyses conducted by Gorbalenya and Koonin (1993) and Singleton et al. (2007) identified three families of helicases within SF1. These families are the UvrD/Rep-like, Pif1-like, and Upf1-like helicases. The UvrD/Rep-like family consists primarily of DNA helicases found in bacteria and a few eukaryotes (Gilhooly et al. 2013). Pif1-like helicases are found in both eukaryotes and prokaryotes. This family includes DNA helicases that promote nuclear and mitochondrial genome stability and DNA repair (Bochman et al. 2010; Gilhooly et al. 2013). SF1 RNA helicases belong to the Upf1-like family. They are conserved RNA helicases that function in nonsense-mediated RNA decay and prevent genome instability during transcription (Mischo et al. 2011; Gupta and Li 2018). These RNA helicases contain sequence domains that are conserved among the Upf1-like RNA helicases and are not found in other RNA helicase families (Fig. 1B). Subdomains 1B and 1C are located in conserved positions within the RNA helicase core (Fairman-Williams et al. 2010). The 1B subdomain forms a β-barrel fold, and 1C forms an α-helical fold within the helicase core, both contributing to modulating conformational changes in the RNA helicase (Gowravaram et al. 2018; Kanaan et al. 2018). Motif IIIa is specific to SF1 RNA helicases and serves as a stacking platform for adenine in ATP through a conserved tyrosine residue (Fairman-Williams et al. 2010). The SF2 superfamily of RNA helicases is the largest and contains ten families of helicases (Fig. 1A). These families were identified by Fairman-Williams et al. (2010) through sequence and phylogenetic analyses. SF2 family members include RecG-like, RecQ-like, Rad3/XPD, DEAD-box, DEAH/RHA, Ski2-like, RIG-I-like, Swi/Snf, NS3/NPH-II, and Type I restriction enzymes. RecG-like helicases are found in prokaryotes, RecQ-like and Rad3/XPD families contain DNA helicases, NS3/NPH-II helicases are viral RNA helicases, and Type I restriction enzymes are part of the restriction–modification system in bacteria. Fungal SF2 RNA helicases are found among the remaining families (Fairman-Williams et al. 2010; Byrd and Raney 2012). 4 IMA Fungus 16: e151785 (2025), DOI: 10.3897/imafungus.16.151785 Amanda M. Seto & Barry J. Saville: U. maydis RNA helicases Figure 1. Helicase superfamilies and characteristics of SF1 and SF2 RNA helicases. A Summary of the six helicase superfamilies found in prokaryotes and eukaryotes; B The characteristics of the N-terminus, C-terminus domains, and RNA helicase core of SF1 and SF2 RNA helicases. The SF1 and SF2 RNA helicases have two RecA-like domains containing specific sequence motifs. These sequence motifs are colored according to their biochemical functions: orange, ATP binding and hydrolysis; blue, RNA binding; and green, coordination between RNA and ATP binding. The N-terminus and C-terminus of RNA helicases contain some conserved domains that are indicated; however, variability in these regions provides specificity to the RNA helicase. The α-8 represents the conserved α-helix at the end of motif II. The triangle represents the position of the β-hairpin structure motif. Abbreviations: HB, helix bundle containing the ratchet helix; HLH, helix-loop-helix; IG, immunoglobulin-like; OB, oligosaccharide-binding fold; WH, winged helix. The distance between domains and sequence motif sizes is not to scale. SF2 SF1 Upf1-like Q1B 1C IIa IV V Vb VI Ib Ic II III Va IIIa RecA-like Domain 1 RNA helicase core RecA-like Domain 2 N-terminus C-terminus DEAH/RHA DEAD-box Ski2-like QIIa IV IVa V Vb VI Ib Ic II III Va IIa IV IVa V Vb VI Ib Ic II III Va WH HB OB QIIa IV IVa V Vb VI Ib Ic II III Va Sec63 α-8 WH HB HLH IG Legend 1B ATP binding & hydrolysis β-barrel fold α-helical fold Winged helix Helix bundle Helix-loop-helix Immunoglobulin-like Oligosaccharide-binding fold RNA binding Coordinates RNA and ATP binding β-Hairpin structure WH OB HLH IG 1C HB Helicase Superfamilies SF1 SF2 SF3 SF4 SF5 SF6 A B AAA+ family of DNA helicases found in prokaryotes and eukaryotes 1. UvrD/Rep-like - primarily bacterial DNA helicases 2. Pif1-like - prokaryote and eukaryote DNA helicases 3. Upf1-like - prokaryote and eukaryote helicases that function on both DNA and RNA Contains three families: Contains ten families: 1. RecG-like - prokaryote helicases 2. RecQ-like - prokaryote and eukaryote DNA helicases 3. Rad3/XPD - prokaryote and eukaryote DNA helicases 4. DEAD-box - primarily prokaryote and eukaryote RNA helicases 5. DEAH/RHA - prokaryote and eukaryote RNA helicases 6. Ski2-like - prokaryote and eukaryote DNA and RNA helicases 7. RIG-I-like - eukaryotic RNA helicases (viral RNA sensors) 8. Swi/Snf - eukaryotic chromatin remodelling helicases 9. NS3/NPH-II - viral RNA helicases 10. Type 1 restriction enzymes - bacterial modification system Contains the bacterial RNA helicase Rho Helicases found in bacteria and bacteriophages Helicases found in DNA and RNA viruses 5 IMA Fungus 16: e151785 (2025), DOI: 10.3897/imafungus.16.151785 Amanda M. Seto & Barry J. Saville: U. maydis RNA helicases The DEAD-box family is the largest family of helicases, with members conserved across prokaryotes and eukaryotes. DEAD-box RNA helicases contain a helicase core composed of 12 sequence motifs. The defining feature of this family is motif II, which contains the amino acid sequence Asp-Glu-Ala-Asp (DEAD) (Fairman-Williams et al. 2010; Jankowsky 2011; Linder and Jankowsky 2011; Byrd and Raney 2012). Many DEAD-box helicases bind to RNA in an ATP-dependent manner. They modulate structured RNAs by disrupting secondary and tertiary structures and can also disrupt RNA–protein interactions in ribonucleoprotein complexes (RNPs). DEAD-box helicases can separate RNA duplexes by disrupting the duplex, thereby accelerating strand separation. This occurs in an ATP-dependent manner but is typically limited to short duplexes (Yang et al. 2007). Structurally, DEAD-box RNA helicases contain an α-helix, termed α-helix 8, located at the end of motif II (Fig. 1B). This structure is proposed to regulate access to the binding site when the helicase is in the ADPbound state. Upon ATP binding, a conformational change occurs that moves α-helix 8 from the binding site to interact with a conserved arginine in motif V (Schütz et al. 2010; Cordin et al. 2012). The DEAH/RNA helicase A (RHA) family contains spliceosomal and RHAgroup subfamilies. Spliceosomal RNA helicases function in pre-mRNA splicing, while RHA-group helicases are involved in transcription, RNA export, and translation. The conserved sequence motifs in DEAH/RHA helicases differ from those in DEAD-box helicases, and DEAD-box helicases have a more variable C-terminal domain (Fig. 1B). In contrast, the C-terminal domain of DEAH/RHA helicases is conserved and includes a winged helix, helix bundle, and oligosaccharide-binding fold. This domain organization contributes to RNA binding strength and the helicase’s translocating activity (De Bortoli et al. 2021). The Q-motif, which is involved in ATP binding, is absent in DEAH/RHA helicases (Fairman-Williams et al. 2010). Structurally, DEAH/RHA helicases contain two β-hairpins (Fig. 1B): one in RecA-like domain 1, between motifs Ib and Ic, and another in RecA-like domain 2, between motifs Vb and VI (Cordin and Beggs 2013; He et al. 2017). The first β-hairpin is an extension of motif Ib, is unique and conserved in DEAH/RHA helicases, and is responsible for unwinding RNA in a 3′-to-5′ direction (He et al. 2017). The β-hairpin in RecA-like domain 2 is longer than that found in the same position in Ski2-like helicases. Structural analyses show that when the helicase is in the ADP state, this β-hairpin blocks access to the nucleic acid binding cavity, which is formed by the two RecA-like domains, the helix bundle, and the winged helix. ATP binding induces a conformational change that removes the β-hairpin from the cavity, allowing RecA-like domain 2 to bind to the 5′ end of the RNA duplex. In the proposed model for strand separation, the β-hairpin slices through the RNA duplex, while the helix bundle pulls the single-stranded RNA through the cavity. The OB-fold is located at the cavity entrance and serves as a docking platform for other binding proteins (He et al. 2010; Walbott et al. 2010; He et al. 2017). The Ski2-like family of SF2 RNA helicases comprises two subfamilies: Ski2 and Brr2. This is a small family with a structure distinct from other SF2 RNA helicases. Like DEAH/RHA helicases, Ski2-like helicases possess a short β-hairpin between motifs Va and VI (Fig. 1B). This β-hairpin is proposed to function similarly to its DEAH/RHA counterpart, assisting in duplex unwinding by positioning itself between the strands (reviewed in Fairman-Williams et al. 2010; 6 IMA Fungus 16: e151785 (2025), DOI: 10.3897/imafungus.16.151785 Amanda M. Seto & Barry J. Saville: U. maydis RNA helicases Johnson and Jackson 2013). At the C-terminus, Ski2-like helicases contain a conserved winged helix and a Sec63 domain (Fig. 1B). The Sec63 domain includes a helix bundle (HB), helix-loop-helix (HLH), and immunoglobulin-like (IG) domains. This domain is unique to the Ski2-like family and may contribute to regulating substrate binding by forming a tunnel through which RNA binds and is translocated during unwinding (Cordin and Beggs 2013). Identification of Ustilago maydis RNA helicases RNA helicases in Ustilago maydis and their putative functions have not been previously investigated. Our interest in the function of U. maydis RNA helicases stemmed from earlier research in our laboratory, which suggested that some gene transcripts are stabilized in the dormant teliospore through the formation of double-stranded RNAs (dsRNAs). During teliospore germination, these stabilized transcripts would need to be unwound and made available for translation (Donaldson and Saville 2013; Ostrowski and Saville 2017). It was proposed that RNA helicases may modulate the availability of mRNAs by unwinding the dsRNAs in the teliospore following the initiation of germination. We identified putative U. maydis RNA helicases and proposed functions based on homology to previously characterized RNA helicases in other model organisms. We then selected RNA helicases with proposed roles in fungal development and growth, inferred from current research in other eukaryotes. Putative U. maydis RNA helicases were identified using previously characterized RNA helicases from Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, and Homo sapiens (Jankowsky 2011; Bourgeois et al. 2016). Known protein sequences of these RNA helicases were compared to those of U. maydis using reciprocal BLASTp, which identified 46 RNA helicases in U. maydis (Table 1). In comparison, approximately 41 RNA helicases have been identified in S. cerevisiae and 70 in H. sapiens (Bohnsack et al. 2023). Plant species such as Arabidopsis thaliana, Zea mays, and Oryza sativa contain more than 150 RNA helicases (Li et al. 2023). The 46 U. maydis RNA helicases were categorized into the SF1 and SF2 superfamilies: four belong to the SF1 Upf1-like group, and 42 are classified within the SF2 superfamily. The SF2 helicases were further divided among the DEAD-box, DEAH/RHA, and Ski2-like families. No U. maydis RNA helicases were identified in the RIG-I-like family, which is expected, as RIG-I-like helicases typically act as viral sensors involved in the antiviral immune response (Byrd and Raney 2012). Additionally, although DICER1 in eukaryotes is classified as a RIG-I-like helicase due to the presence of a helicase domain (Table 1), an ortholog in U. maydis was not expected, as this species lacks the RNA interference (RNAi) machinery (Nakayashiki et al. 2006). Further classification of U. maydis RNA helicases was carried out through phylogenetic analyses using protein sequences from U. maydis, Sporisorium reilianum, Ustilago hordei, S. cerevisiae, Schizosaccharomyces pombe, Neurospora crassa, C. elegans, H. sapiens, Dictyostelium discoideum, and A. thaliana. Protein sequences were aligned using Jalview v2.11 (Waterhouse et al. 2009) and the JABAWS service to perform MUSCLE alignment under default settings (Edgar 2004; Troshin et al. 2011). Maximum likelihood phylogenetic trees were then constructed for each RNA helicase family (Figs 2–5). 7 IMA Fungus 16: e151785 (2025), DOI: 10.3897/imafungus.16.151785 Amanda M. Seto & Barry J. Saville: U. maydis RNA helicases Table 1. RNA helicases in U. maydis and their orthologs in S. cerevisiae, C. elegans, D. melanogaster, M. musculus, and H. sapiens. Superfamily Family U. maydis S. cerevisiae C. elegans D. melanogaster M. musculus H. sapiens SF1 Upf1-like UMAG_10602 Sen1 eri-7 CG7504 Setx SETX UMAG_11428 Upf1 smg-2 Upf1 Upf1 UPF1 UMAG_01122 Hcs1 Y106G6D.5 IGHMBP2 IGHMBP2 UMAG_10130 CG6701 MOV10 MOV10 Ecm32 armi Mov10l1 MOV10L1 C44H9.4 Helz Helz HELZ Helz2 HELZ2 emb-4 CG14729 Aqr AQR SF2 DEAD-box UMAG_05482 Tif1 inf-1 eIF4A Ddx2a DDX2A Tif2 F57B9.3 Ddx2b DDX2B UMAG_06129 Fal1 F33D11.10 Y65B4A.6 eIF4A-III DDX48 DDX48 UMAG_10655 Dhh1 cgh-1 me3B DDX6 DDX6 mel-46 Gem3 DDX20 DDX20 UMAG_03765 Dbp5 ddx-19 DBP80 DDX19a DDX19A DDX19b DDX19B UMAG_11769 Sub2 hel-1 Hel25E DDX39A DDX39A DDX39B DDX39B UMAG_05873 UMAG_10410 Has1 B0511.6 CG6375 DDX18 DDX18 UMAG_11989 Dbp4 ddx-10 CG5800 DDX10 DDX10 Y55F3BR.1 CG9054 DDX1 DDX1 UMAG_03268 Spb4 ZK512.2 DDX55 DDX55 DDX55 UMAG_06228 Dbp7 CG8611 DDX31 DDX31 UMAG_05214 Rrp3 T26G10.1 CG9253 DDX47 DDX47 UMAG_10241 Dbp8 H20J04.4 DBP45A DDX49 DDX49 UMAG_03170 Drs1 ddx-27 Rs1 DDX27 DDX27 UMAG_05200 Dbp10 Y94H6A.5 CG32344 DDX54 DDX54 UMAG_04080 Ded1 laf-1 Belle DDX3X DDX3X Dbp1 vbh-1 DDX3Y DDX3Y D1Pas1 glh-1 Vasa DDX4 DDX4 glh-2 UMAG_10095 Dbp2 ddx-17 Rm62 DDX5 DDX5 DDX17 DDX17 CG7878 DDX53 DDX43 DDX43 UMAG_01732 Dbp3 UMAG_04587 sacy-1 abs DDX41 DDX41 UMAG_10666 Prp28 ddx-23 CG10333 DDX23 DDX23 C46F11.4 DmRH27 DDX42 DDX42 UMAG_01174 Prp5 ddx-46 CG6227 DDX46 DDX46 UMAG_10683 Rok1 ddx-52 DmRH17 DDX52 DDX52 UMAG_03892 Dbp9 C24H12.4 CG1666 DDX56 DDX56 UMAG_00921 Dbp6 ZK686.2 Dbp73D DDX51 DDX51 Ddx21 DDX21 8 IMA Fungus 16: e151785 (2025), DOI: 10.3897/imafungus.16.151785 Amanda M. Seto & Barry J. Saville: U. maydis RNA helicases Superfamily Family U. maydis S. cerevisiae C. elegans D. melanogaster M. musculus H. sapiens SF2 DEAD-box Ddx50 DDX50 UMAG_00242 Mak5 F55F8.2 DDX24 DDX59 DDX59 UMAG_00652 Mss116 UMAG_06314 Mrh4 Dbp21E2 DDX28 DDX28 UMAG_00835 DEAH/RHA UMAG_10915 Prp2 mog-4 CG10689 DHX16 DHX16 UMAG_04188 Prp16 mog-1 CG32604 DHX38 DHX38 UMAG_03936 Prp22 mog-5 CG8241 DHX8 DHX8 UMAG_11281 Prp43 ddx-15 CG11107 DHX15 DHX15 DHX32 DHX32 UMAG_00419 Dhr2 let-355 DHX33 UMAG_11913 ddx-35 DHX35 UMAG_00574 CG9323 DHX29 DHX29 CG1582 DHX57 DHX57 UMAG_11114 YLR419W UMAG_05767 rha-1 mle DHX9 DHX9 DHX30 DHX36 DHX36 UMAG_04665 Dhr1 rha-2 kurz DHX37 DHX37 smgl-2 CG32533 DHX34 DHX34 DHX40 DHX40 Ski2-like UMAG_00393 Ski2 skih-2 tst SkiV2l SKIV2L UMAG_11667 Mtr4 mtr-4 mtr4 SKIV2L2 MTREX UMAG_03738 Brr2 snrp-200 CG5931 Snrnp200 SNRNP200 UMAG_00282 Slh1 Y54E2A.4 obe Ascc3 ASCC3 UMAG_04997 Suv3 C08F8.2 Suv3 Supv3l1 SUPV3L1 RIG-I-like C28H8.3 DDX60 DDX60 dcr-1 dcr1 Dicer1 DICER1 drh-1 DDX58 DDX58 Ifih1 IFIH1 DHX58 There are four SF1 Upf1-like RNA helicases in S. cerevisiae and eight in H. sapiens (Table 1). Our analysis identified four Upf1-like RNA helicases in U. maydis. There are three putative orthologs of the characterized SF1 RNA helicases Sen1 (UMAG_10602), Upf1 (UMAG_11428), and Hcs1 (UMAG_01122). Phylogenetic analysis identified the remaining RNA helicase as a putative basidiomycete-specific SF1 RNA helicase (UMAG_10130) (Fig. 2). Of the 42 SF2 RNA helicases, our investigation identified 27 U. maydis DEADbox RNA helicases (Table 1), of which 25 are conserved and have orthologs in other eukaryotes (Fig. 3). In S. cerevisiae, there are 26 DEAD-box RNA helicases, compared to 33 in H. sapiens. The remaining two DEAD-box RNA helicases in U. maydis (UMAG_05873 and UMAG_00835) are basidiomycete-specific (Fig. 3). Many of these RNA helicases function in a specific aspect of RNA metabolism; however, a few are involved in multiple areas of RNA metabolism. A total of 10 DEAH/RHA RNA helicases were identified in U. maydis based on our sequence (Table 1) and phylogenetic (Fig. 4) analyses, compared to seven in S. cerevisiae and 15 in H. sapiens. Of the 10, six are spliceosomal DEAH 9 IMA Fungus 16: e151785 (2025), DOI: 10.3897/imafungus.16.151785 Amanda M. Seto & Barry J. Saville: U. maydis RNA helicases RNA helicases, and the remaining four are RHA-group RNA helicases. The phylogenetic tree (Fig. 4) suggests the presence of a fungal-specific DEAH/RHA RNA helicase with no orthologs in the eukaryotes included in our phylogenetic analysis. This fungal-specific RNA helicase was identified as YLR419W in S. cerevisiae and UMAG_11114 in U. maydis. The last family of SF2 RNA helicases is the Ski2-like RNA helicases. Data from S. cerevisiae identifies four Ski2-like SF2 RNA helicases: Brr2, Slh1, Mtr4, and Ski2. Suv3 was originally categorized as Ski2-like but has since been removed following further structural analysis (reviewed in Johnson and Jackson 2013). Ustilago maydis has orthologs to all five Ski2-like RNA helicases (Fig. 5). Functions of RNA helicases and their potential roles in Ustilago maydis RNA helicases function in all aspects of RNA metabolism, with demonstrated roles in transcription, pre-mRNA splicing, ribosome biogenesis, RNA export, translation, and RNA degradation (Bleichert and Baserga 2007; Jankowsky 2011; Linder and Jankowsky 2011). Despite their broad roles in RNA metabolism, some RNA helicases have specific functions, while others are involved in multiple processes (Jankowsky 2011). Several studies in H. sapiens show that upregulation or downregulation of RNA helicases contributes to the development of various diseases Figure 2. Maximum likelihood phylogenetic tree of SF1 RNA helicases. The phylogenetic tree was created with orthologs from U. maydis, S. reilianum, U. hordei, C. neoformans, S. cerevisiae, S. pombe, N. crassa, A. thaliana, C. elegans, D. discoideum, and H. sapiens using W-IQ-TREE multicore version 1.6.12 with default settings (Trifinopoulos et al. 2016), 1,000 ultrafast bootstrap alignments, and the approximate Bayes test. The tree was visualized with FigTree v1.4.4 (http://tree.bio. ed.ac.uk/software/figtree/), rooted at the midpoint, and bootstrap values are indicated for each node. Phylogenetic groups containing an RNA helicase ortholog in U. maydis were collapsed and color-coded. The U. maydis RNA helicase and the number of sequences for each clade are identified for each collapsed group. Each collapsed group was named after the S. cerevisiae and H. sapiens orthologs. The scale bar indicates the expected number of substitutions per amino acid. The original phylogenetic tree is in Suppl. material 1: fig. S1 and contains all gene names and organisms used in this analysis. 0.9 MOV-10 [Homo sapiens] SDE3 [Arabidopsis thaliana] HELZ2 [Homo sapiens] Ecm32 [Saccharomyces cerevisiae] HELZ [Homo sapiens] 100 61 77 100 98 58 81 100 100 99 81 Hcs1/IGHMBP2 Sen1/SETX Upf1/UPF1 Basidiomycete-specific UMAG_01122 (n = 12) UMAG_10602 (n = 10) UMAG_10130 (n = 4) UMAG_11428 (n = 11) 16 IMA Fungus 16: e151785 (2025), DOI: 10.3897/imafungus.16.151785 Amanda M. Seto & Barry J. Saville: U. maydis RNA helicases Table 4. Functions of RNA helicases during pre-mRNA splicing. Orthologs for U. maydis, S. cerevisiae, and H. sapiens are listed. Superfamily Family RNA helicase orthologs RNA helicase function during premRNA splicing References U. maydis S. cerevisiae H. sapiens SF2 DEAD-box UMAG_11769 Sub2 DDX39A DDX39B Responsible for the displacement and recruitment of specific RNPs at the premRNA intron branch site; DDX39A and DDX39B regulate alternative splicing Luo et al. (2001); Liu and Cheng (2015); Banerjee et al. (2024) UMAG_01174 Prp5 DDX46 Interacts with snRNP U2 to mediate conformation changes and stabilization; Release of Prp5 signals the recruitment of the U4/U6.U5 tri-snRNP; DDX46 induces pre-mRNA conformational changes for splicing Liu et al. (2007); Liang and Cheng (2015); Bourgeois et al. (2016) UMAG_10666 Prp28 DDX23 Destabilizes the interaction between snRNP U1 and the 5’ splice site and allows for U6 to bind to the site; May proofread the 5’ splice site during spliceosome assembly Liu and Cheng (2015); Bourgeois et al. (2016) UMAG_04188 Prp16 DDX38 Induces spliceosome conformational changes before initiation of the second catalytic reaction; Displaces proteins associated with the spliceosome causing the catalytic core to be less rigid to allow for contact with the 3’ splice site; Prp16 functions with Prp22 to remove stalled spliceosomes and repress suboptimal splicing sites to allow for alternative splice site selection Liu and Cheng (2015); Robert-Paganin et al. (2015); Bourgeois et al. (2016); Semlow et al. (2016) UMAG_10095 Dbp2 DDX5 DDX17 DDX5 mediates the interaction between U1 and the 5’ splice site; Serves as a bridge for communication between the splice site and the U4/U6.U5 tri-snRNP DDX17 assists the U1 in recognizing the 5’ splice site and facilitates alternative splicing in select human genes Hönig et al. (2002); Lee (2002); Liu (2002) UMAG_04080 Ded1 Dbp1 DDX3 Interacts with the spliceosome however exact role is not fully understood Jamieson et al. (1991) UMAG_04587 DDX41 DDX41 and the C. elegans ortholog sacy-1 interact with spliceosome components and can affect alternative splicing Polprasert et al. (2015); Tsukamoto et al. (2020); Andreou (2021) UMAG_06129 Fal1 DDX48 DDX48 facilitates the assembly of the exon junction complex Linder and Jankowsky (2011) UMAG_5200 Dbp10 DDX54 DDX54 binds to introns that contain a weak 3’ splice site to increase the splicing rate Rodríguez-Galán et al. (2013); Milek et al. (2017) DEAH/ RHA UMAG_10915 Prp2 DHX16 Remodels the spliceosome into its catalytically active form with cofactor Spp2; ATPase activity destabilizes the snRNPU2 from the spliceosome to expose the branchpoint Liu and Cheng (2015); Bourgeois et al. (2016) UMAG_03936 Prp22 DHX8 Facilitates the release of mature mRNA by disrupting RNA-RNA or RNA-protein interactions; Capable of discarding aberrant spliceosomes from pre-mRNA; Prp22 and Prp16 function together to repress suboptimal splicing sites and aid in alternative splice site selection Robert-Paganin et al. (2015); Semlow et al. (2016) UMAG_11281 Prp43 DHX15 Mediates disassembly of the spliceosome after splicing completion or removal of an impaired or arrested spliceosome Liu and Cheng (2015) 17 IMA Fungus 16: e151785 (2025), DOI: 10.3897/imafungus.16.151785 Amanda M. Seto & Barry J. Saville: U. maydis RNA helicases UMAG_04587 is a putative RNA helicase with orthologs in basidiomycete fungi and animals; however, no orthologs were identified in the ascomycete fungi included in our analysis (Fig. 3). STRING analysis suggests that UMAG_04587 interacts with proteins involved in pre-mRNA splicing (Suppl. material 2: table S1), consistent with other DDX41 orthologs. In H. sapiens, DDX41 may modulate the expression of a subset of proteins at the posttranslational level (Peters et al. 2017). In D. melanogaster, the ortholog Abstrakt is involved in the posttranscriptional regulation of the gene Insc by binding to its mRNA. Loss of Abstrakt results in decreased Insc protein levels and defects in cell polarity and division (Irion et al. 2004). DDX41 also plays a role in the innate immune response and acts as an antiviral factor against viral infections by sensing viral DNA and DNA:RNA duplexes, thereby modulating nucleic acids to trigger a host immune response (Li et al. 2018; Soponpong et al. 2018; Liu et al. 2019; Winstone et al. 2024). If UMAG_04587 functions as a viral sensor, it may contribute to detecting and responding to mycovirus infections. The presence of its transcript in the cyan/tumor module during pathogenesis (Lanver et al. 2018; Table 2) suggests that UMAG_04587 may play an enhanced role during the onset of tumor formation in planta, possibly by modulating gene expression at the posttranscriptional or posttranslational level. We hypothesize that if UMAG_04587 regulates cell polarity during growth, deletion mutants will exhibit growth defects. STRING analysis predicts that UMAG_10666 interacts with other spliceosomal proteins, suggesting a functional role in pre-mRNA splicing in U. maydis (Suppl. material 2: table S1). This RNA helicase is the ortholog of Prp28 in S. cerevisiae and DDX23 in H. sapiens (Fig. 3 and Table 1). The C. elegans ortholog DDX-23 is required for embryonic and postembryonic development and cell differentiation, possibly by modulating specific RNA-protein interactions (Konishi et al. 2008; Chu et al. 2016). If this function is conserved, UMAG_10666 may regulate a specific subset of genes during key developmental transitions. Its transcript profile during pathogenesis suggests that elevated levels of UMAG_10666 may be required for splicing transcripts involved in the early stages of pathogenesis and/or in modulating RNA-protein interactions and splicing of genes that drive tumor formation. UMAG_01174 was identified as the putative ortholog to Prp5/DDX46 (Fig. 3). STRING analysis predicts that UMAG_01174 interacts with proteins involved Superfamily Family RNA helicase orthologs RNA helicase function during premRNA splicing References U. maydis S. cerevisiae H. sapiens SF2 DEAH/ RHA UMAG_05767 DHX9 Binds to pre-mRNA and snRNPs that are components of the splicing machinery suggesting a possible role during splicing Lee and Pelletier (2016) UMAG_11913 DHX35 DHX35 interacts with other spliceosome proteins Sales-Lee et al. (2021) Ski2-like UMAG_03738 Brr2 SNRNP200 Brr2 is a component of the snRNP U5 and mediates the unwinding of U4/ U6 to release U4 during spliceosome activation; Regulates other splicing factors by serving as a platform for their recruitment; Promotes protein-protein interactions; Capable of inducing structural changes to the pre-mRNA Liu and Cheng (2015) 18 IMA Fungus 16: e151785 (2025), DOI: 10.3897/imafungus.16.151785 Amanda M. Seto & Barry J. Saville: U. maydis RNA helicases in pre-mRNA splicing (Suppl. material 2: table S1). Interestingly, STRING also predicts an interaction with the uncharacterized protein UMAG_11076. This protein is the putative ortholog of the S. cerevisiae protein Urn1, which plays a role in cell cycle progression (Niu et al. 2008). Similarly, DDX46 has been shown to contribute to cell proliferation and cell cycle progression pathways (Li et al. 2016). If the role of this RNA helicase is conserved across species, UMAG_01174 may contribute to cell cycle progression and proliferation in U. maydis cells. This helicase is also present in the cyan/tumor module during pathogenesis (Lanver et al. 2018) (Table 2), suggesting that its activity may modulate the splicing of transcripts encoding proteins or directly affect proteins that contribute to U. maydis growth and tumor induction. The putative SF2 RNA helicase UMAG_11913 was identified as the ortholog of DHX35 in H. sapiens (Fig. 4, Table 1). STRING analysis suggests that UMAG_11913 interacts with other spliceosomal proteins, further supporting a conserved function across species (Suppl. material 2: table S1). Functional characterization of UMAG_11913 may confirm its role in the U. maydis spliceosome. In Magnaporthe oryzae, another fungal plant pathogen, MoDHX35 deletion mutants exhibit reduced appressorium formation and decreased virulence (Ying et al. 2022). The UMAG_11913 transcript is found in the light-cyan module during U. maydis pathogenesis, indicating that it is upregulated during the early stages of infection, followed by a decrease and then an increase (Lanver et al. 2018). We hypothesize that if the molecular regulation of UMAG_11913 mirrors that of MoDHX35, appressorium formation in U. maydis may be impaired, leading to reduced pathogenicity. Deletion strains of UMAG_11913 should be generated to assess whether a shared phenotype exists with M. oryzae. A similar phenotype would suggest a conserved function for this RNA helicase in fungal plant pathogens. Fal1, the budding yeast ortholog of UMAG_06129, is essential in S. cerevisiae, whereas deletion mutants are viable in fission yeast (Kressler et al. 1997; Kim et al. 2010). Our STRING analysis of UMAG_06129 (Suppl. material 2: table S1) predicts protein-protein interactions with components of the splicing machinery and ribosome biogenesis. Interestingly, S. pombe Fal1 interacts with Red5, a subunit of the MTREC complex, to regulate the splicing of meiosis transcripts. These transcripts are unspliced and targeted for degradation during mitosis. The interaction between Fal1 and Red5 enables splicing of meiosis-specific genes during meiosis (Marayati et al. 2016). The U. maydis genome contains orthologs of Red5, most MTREC complex components, and the EJC, suggesting that UMAG_06129 may function similarly by identifying meiosis transcripts and facilitating their translation during meiosis. The UMAG_06129 transcript is upregulated in the dormant teliospore compared to the haploid and dikaryon cell types (Seto et al. 2025). It is the only RNA helicase in the salmon module, which includes genes upregulated after tumor formation and during teliospore development (Table 2) (Lanver et al. 2018). This expression pattern suggests a role in the regulation of genes associated with teliospore development, dormancy entry, and meiotic pause. UMAG_06129 may contribute to the translation of these genes and the early meiotic processes before teliospore dormancy. Functional characterization of UMAG_06129 will clarify its role in U. maydis. If it functions similarly to its S. pombe ortholog, it may influence teliospore formation and germination, processes that coincide with meiosis in U. maydis. 19 IMA Fungus 16: e151785 (2025), DOI: 10.3897/imafungus.16.151785 Amanda M. Seto & Barry J. Saville: U. maydis RNA helicases RNA export After splicing, the mRNA undergoes additional modifications and binds various proteins to become competent for nuclear export. This process requires the recruitment of multiple adaptor proteins, which depend on RNA helicases to facilitate recruitment, protein dissociation, or mRNA remodeling in both the nucleus and cytoplasm (Iglesias and Stutz 2008; Bourgeois et al. 2016). The RNA helicases Sub2, Dbp2, and Dbp5 in S. cerevisiae have been identified as playing roles in RNA export from the nucleus, and their U. maydis orthologs are UMAG_11769, UMAG_10095, and UMAG_03765, respectively (Table 5). Transcriptome analysis of the three U. maydis RNA helicases predicted to be involved in RNA export revealed that both UMAG_11769 and UMAG_03765 have transcript levels that are not significantly different between cell types or during teliospore dormancy and germination (Donaldson et al. 2017; Seto et al. 2025). Both transcripts are also found in the yellow module during pathogenesis, indicating co-expression with genes involved in cellular metabolism (Table 2) (Lanver et al. 2018). The UMAG_10095 transcript was found to be upregulated in the dormant teliospore and remained unchanged during teliospore germination (Seto et al. 2025). During pathogenesis, UMAG_10095 is present in the green-yellow module (Table 2) (Lanver et al. 2018), representing gene transcripts that increase and peak at 2 dpi before decreasing during the late stages of pathogenesis. UMAG_11769 and UMAG_10095 were identified as having possible roles in U. maydis growth, pathogenesis, and regulation of teliospore dormancy and germination. UMAG_11769 was previously described by Feldbrügge et al. (2008) as the putative ortholog to the S. cerevisiae RNA helicase Sub2 and H. sapiens DDX39A/ DDX39B (Fig. 3, Table 1). STRING analysis predicts protein-protein interactions with proteins involved in spliceosome assembly and the export of polyadenylated mRNAs (Suppl. material 2: table S1), suggesting a conserved function across eukaryotes. The Danio rerio ortholog, Ddx39ab, is required for the development of the heart, trunk muscles, and eyes during embryogenesis. This helicase binds to a specific subset of epigenetic regulatory factors involved in the development of these tissues. Loss of ddx39ab results in abnormal pre-mRNA splicing and misregulation of developmental genes (Zhang et al. 2018). The H. sapiens orthologs DDX39A and DDX39B (Fig. 3, Table 1) are components of the mRNA export Table 5. Functions of RNA helicases during RNA export. Orthologs in U. maydis, S. cerevisiae, and H. sapiens are listed. Superfamily Family RNA helicase orthologs RNA helicase function during RNA export References U. maydis S. cerevisiae H. sapiens SF2 DEAD-box UMAG_11769 Sub2 DDX39A DDX39B Responsible for recruiting the protein Yra1 to the mRNA and is then displaced to allow the mRNP to dock onto the nuclear pore complex Luo et al. (2001); Sträßer and Hurt (2001); Noble et al. (2011) UMAG_10095 Dbp2 DDX5 DDX17 Aids in the assembly of Yra1, Nab2, and Mex67 on the poly(A)+ tail by unwinding and remodelling the mRNA duplex Ma et al. (2013); Xing et al. (2019) UMAG_03765 Dbp5 DDX19A DDX19B DDX25 Dbp5 initiates mRNA remodelling when an mRNP is exported through the nuclear pore Tran et al. (2007); Noble et al. (2011) 20 IMA Fungus 16: e151785 (2025), DOI: 10.3897/imafungus.16.151785 Amanda M. Seto & Barry J. Saville: U. maydis RNA helicases machinery and are linked to mitotic progression. It has been proposed that these helicases regulate gene expression of mitosis-related genes, with each helicase targeting different gene sets (Yamazaki et al. 2010). If UMAG_11769 functions similarly, we predict that deletion mutants will exhibit growth and mitotic defects. STRING analysis for the Dbp2 ortholog UMAG_10095 predicts interactions with proteins involved in transcription, splicing, ribosome biogenesis, RNA export, and RNA degradation (Suppl. material 2: table S1), supporting a conserved function. In yeast and mammals, Dbp2/DDX5 is linked to maintaining cellular homeostasis. It plays a role in glucose sensing and promoting glycolysis during growth (Beck et al. 2014; Xing et al. 2017). More recently, Dbp2 has been shown to modulate the stress response to low glucose by binding specific mRNAs, retaining them in the nucleus, and promoting their degradation under nutrient-rich conditions. Under nutrient-poor conditions, Dbp2 relocates to the cytoplasm to facilitate export of these mRNAs for translation (Paul et al. 2025). We previously hypothesized that upregulation of UMAG_10095 in dormant and germinating teliospores enables U. maydis to detect changes in glucose availability and coordinate RNA processing accordingly (Seto et al. 2025). DBP2 is nonessential in S. cerevisiae (Bond et al. 2001; Cloutier et al. 2012), and we predict that UMAG_10095 deletion will be nonlethal in U. maydis, but deletion or overexpression mutants will likely exhibit a slow growth phenotype, as observed in S. cerevisiae DBP2 mutants (Cloutier et al. 2012). Additionally, UMAG_10095 deletion mutants may be impaired in teliospore germination. Ribosome biogenesis Approximately 20 different RNA helicases are involved in ribosome biogenesis, with each helicase performing a specific function during the process. The U. maydis orthologs of these RNA helicases are listed in Table 6. The first step of ribosome biogenesis is the transcription of rRNA. The large primary rRNA transcript—called the 35S pre-rRNA in S. cerevisiae—is transcribed by RNA polymerase I and contains the 18S, 5.8S, and 25S/28S rRNAs. The 5S rRNA is transcribed separately by RNA polymerase III. Processing of the pre-rRNA involves cleavage at specific sites in the external and internal transcribed spacers (ETS and ITS) by endonucleases and exonucleases, producing mature rRNAs (Martin et al. 2013; Rodríguez-Galán et al. 2013). The ribosome is composed of the large and small subunits. The large subunit, known as the 60S in eukaryotes, consists of the 25S/28S, 5.8S, and 5S rRNAs and 46 ribosomal proteins (Martin et al. 2013). The SF2 DEAD-box RNA helicases involved in processing the 60S subunit in S. cerevisiae include Dbp2, Dbp3, Dbp6, Dbp7, Dbp9, Dbp10, Drs1, Mak5, Mtr4, and Spb4. The 40S, the small ribosomal subunit in eukaryotes, comprises the 18S rRNA and 33 other ribosomal proteins. Seven different RNA helicases are involved in processing the 40S subunit: Dbp4, Dbp8, Dhr1, Dhr2, Fal1, Rok1, and Rrp3. Two RNA helicases, Prp43 and Has1, participate in processing both the 60S and 40S subunits (Martin et al. 2013; Rodríguez-Galán et al. 2013). It has also been suggested that Ded1 in S. cerevisiae may function during ribosome biogenesis, as the protein has been detected in association with pre-ribosomal particles, although its exact role is not currently understood (Krogan et al. 2004; Sharma and Jankowsky 2014). Table 6 summarizes the specific functions of these RNA helicases during ribosome biogenesis. 21 IMA Fungus 16: e151785 (2025), DOI: 10.3897/imafungus.16.151785 Amanda M. Seto & Barry J. Saville: U. maydis RNA helicases Table 6. Functions of RNA helicases during ribosome biogenesis. Orthologs in U. maydis, S. cerevisiae, and H. sapiens are listed. Superfamily Family RNA helicase orthologs RNA helicase function during ribosome biogenesis References U. maydis S. cerevisiae H. sapiens SF2 DEAD-box UMAG_10095 Dbp2 DDX5 DDX17 Dbp2 facilitates structural rearrangements of the pre-rRNA; DDX5 and DDX17 are capable of displacing snoRNA U8 from the pre-rRNA Bond et al. (2001); Jalal et al. (2007) UMAG_01732 Dbp3 Facilitate access to the A3 cleavage site of ITS1 in the 27S A2 pre-rRNA for the RNase MRP complex Weaver et al. (1997) UMAG_00921 Dbp6 DDX51 Dbp6 facilitates structural rearrangements of the pre-rRNA to allow for efficient assembly of the 60S ribosomal subunit DDX51 promotes the displacement of proteins during the processing of the 3’ end of the 28S rRNA Kressler et al. (1998); Srivastava et al. (2010); Martin et al. (2013); Rodríguez-Galán et al. (2013) UMAG_06228 Dbp7 DDX31 Dbp7 remodels the 35S pre-rRNA which allows for the attachment and release of proteins during the maturation of the prerRNA Fukawa et al. (2012); Aquino et al. (2021) DDX31 regulates rRNA transcription UMAG_03892 Dbp9 DDX56 May modulate pre-rRNA rearrangements during 18S rRNA maturation Zirwes et al. (2000); Daugeron et al. (2001); RodríguezGalán et al. (2013) DDX56 may function during the maturation of pre-rRNA during maturation of the 60S ribosomal subunit UMAG_5200 Dbp10 DDX54 Bind to pre-rRNA to induce conformational changes or act as a chaperone for other proteins to facilitate cleavage Burger et al. (2000); Rodríguez-Galán et al. (2013); Mitterer et al. (2023) UMAG_03170 Drs1 DDX27 Drs1 is tightly associated with Dbp6, Dbp7, Dbp9, Mak5, and Has1 during synthesis of the 60S ribosomal subunit DDX27 associates with pre-rRNA to recruit the protein complex PeBoW Ripmaster et al. (1992); Bernstein et al. (2006); Martin et al. (2013); Kellner et al. (2015) UMAG_00242 Mak5 DDX24 Mak5 may function within a protein cluster or creates a stable association between ribosomal proteins during 60S assembly DDX24 interacts with MDM2 to mediate ubiquitylation and degradation of p53 to increase transcription of the 47S transcript Zagulski et al. (2003); Pratte et al. (2013); Yamauchi et al. (2014) UMAG_11667 Mtr4 MTREX Functions within the TRAMP complex that contributes to the polyadenylation of 3’ ends of snoRNAs and rRNAs by modulating the activity of the TRAMP complex by unwinding the RNA to generate longer stretches of ssRNA; Can impact RNA binding, ATP affinity, rate of adenylation, and TRAMP dissociation Jia et al. (2011); Martin et al. (2013); Rodríguez-Galán et al. (2013) UMAG_03268 Spb4 DDX55 Spb4 interacts with Pwp2 to facilitate the processing of the 35S pre-rRNA during the early and late stages of maturation DDX55 remodels pre-rRNA during the maturation process Sachs and Davis (1990); de la Cruz et al. (1998a); Dosil and Bustelo (2004); García-Gómez et al. (2011); Choudhury et al. (2021) UMAG_11989 Dbp4 DDX10 May facilitate the release of snoRNAs from pre-rRNA Savitsky et al. (1996); Koš and Tollervey (2005) 22 IMA Fungus 16: e151785 (2025), DOI: 10.3897/imafungus.16.151785 Amanda M. Seto & Barry J. Saville: U. maydis RNA helicases Superfamily Family RNA helicase orthologs RNA helicase function during ribosome biogenesis References U. maydis S. cerevisiae H. sapiens SF2 DEAD-box UMAG_10241 Dbp8 DDX49 Dbp8 is involved in the maturation of the 18S rRNA Daugeron and Linder (2001); Granneman et al. (2006); Awasthi et al. (2018) DDX49 binds to regulatory regions to provide stability to the rRNA UMAG_06129 Fal1 DDX48 Fal1 may function during the maturation of the 18S rRNA Kressler et al. (1997); Alexandrov et al. (2011); Rodríguez-Galán et al. (2013); Choe et al. (2014) DDX48 may remodel or promote unwinding when a secondary structure is encountered UMAG_10683 Rok1 DDX52 Facilitates the release of snoRNAs from pre-rRNA which allows access to cleavage sites Venema et al. (1997); Bohnsack et al. (2008); Martin et al. (2013); Rodríguez-Galán et al. (2013) UMAG_05214 Rrp3 DDX47 Mediates structural changes that allow for snoRNAs to bind and cleavage to the pre-rRNA O’Day et al. (1996); Sekiguchi et al. (2006); Martin et al. (2013); RodríguezGalán et al. (2013) UMAG_10410 Has1 DDX18 Coordinates the assembly of proteins on the pre-rRNA to allow for cleavage to occur Martin et al. (2013); Rodríguez-Galán et al. (2013) UMAG_04080 Ded1 Dbp1 DDX3 Protein detected with pre-ribosomal particles, but function is unknown Krogan et al. (2004); Sharma and Jankowsky (2014) DEAH/ RHA UMAG_04665 Dhr1 DHX37 Remodels pre-rRNA for processing and releases U3 to allow for cleavage to occur in the 5’ ETS and ITS1 Colley et al. (2000); Choudhury et al. (2019) UMAG_ 00419 Dhr2 DHX32 Dhr2 initiates cleavage at site A0 of the 5’ ETS region of the pre-rRNA Colley et al. (2000); Choque et al. (2011); Martin et al. (2013) DHX32 interacts with pre-rRNA processing proteins during rRNA maturation UMAG_11281 Prp43 DHX15 May displace snoRNAs from the pre-rRNA and modulate structural changes to the transcript Rodríguez-Galán et al. (2013) Many RNA helicases involved in ribosome biogenesis are essential proteins. We used current knowledge and U. maydis transcriptome data to identify nine RNA helicases with potential roles in the U. maydis life cycle. The UMAG_11989, UMAG_30170, UMAG_10683, and UMAG_11281 transcripts were not significantly different across all cell types and during teliospore dormancy and germination (Donaldson et al. 2017; Seto et al. 2025). During pathogenesis, these transcripts are found in the green-yellow (UMAG_11989, UMAG_30170, UMAG_10683) and green (UMAG_11281) modules (Table 2). The transcripts for UMAG_10410, UMAG_05214, UMAG_01732, UMAG_10241, and UMAG_05200 were upregulated in the dormant teliospore compared to the haploid and dikaryon cell types (Seto et al. 2025). These transcripts are present in the green (UMAG_05214), green-yellow (UMAG_10410, UMAG_10241), yellow (UMAG_01732), and magenta (UMAG_05200) modules during pathogenesis (Table 2). We predict that these RNA helicases contribute to U. maydis growth, pathogenesis, stress response, and teliospore dormancy. 23 IMA Fungus 16: e151785 (2025), DOI: 10.3897/imafungus.16.151785 Amanda M. Seto & Barry J. Saville: U. maydis RNA helicases The U. maydis ortholog of the S. cerevisiae RNA helicase Has1 was identified as UMAG_10410 (Fig. 3, Table 1). Based on STRING analysis, its role in rRNA processing is likely conserved (Suppl. material 2: table S1). Has1 is essential in S. cerevisiae, and we predict that deletion of UMAG_10410 will be lethal. In zebrafish, the ortholog DDX18 is involved in cell cycle progression during embryogenesis; loss of this RNA helicase results in cell cycle arrest and disrupted hematopoiesis (Payne et al. 2011). In human lung cancer cells, DDX18 depletion causes cell cycle arrest at the G1 phase, whereas overexpression promotes cell proliferation (Feng et al. 2024). We predict that altering the expression of UMAG_10410 will result in growth defects. Based on the U. maydis pathogenesis transcriptome data from Lanver et al. (2018), the peak transcript level of UMAG_10410 at 2 dpi corresponds to the shift to cell proliferation following cell cycle arrest before appressoria penetration. This pattern suggests that UMAG_10410 may play a role during key developmental transitions in U. maydis growth within the plant. Additionally, UMAG_10410 is upregulated in the dormant teliospore and remains stably expressed during teliospore germination (Seto et al. 2025). This expression pattern indicates that UMAG_10410 may have an important role during the transition from dormancy to germination, possibly through maintaining genome stability or regulating DNA repair during this developmental switch. UMAG_11989 is the ortholog to S. cerevisiae RNA helicase DBP4 and DDX10 in H. sapiens. Overexpression of the H. sapiens ortholog DDX10 results in the proliferation of several types of cancer (Yassin et al. 2010; Zhou et al. 2022). In colorectal cancer cells, DDX10 was shown to interact with the ribosomal protein RPL35, which is a component of the 60S subunit. Overexpression of this RNA helicase resulted in alternative splicing of RPL35 mRNA, which then affected the downstream E2F pathway—a pathway that regulates the cell cycle and cancer development (Zhou et al. 2022). Ustilago maydis contains the ortholog to RPL35 (UMAG_11625), indicating the potential for UMAG_11989 to function similarly. Based on U. maydis transcriptome data, UMAG_11989 may have an enhanced role during the early stages of pathogenesis, possibly during the establishment of biotrophic development after the dikaryotic filaments penetrate the plant host. It is hypothesized that it enhances the expression of a subset of genes required for cell cycle progression. If UMAG_11989 regulates the cell cycle through its interaction with other proteins, altering its expression may affect the growth of U. maydis cells and impact its development within plant tissue. In H. sapiens, cell homeostasis can be disrupted by unscheduled R-loop formation. The RNA helicase DDX47 resolves R-loop formation by unwinding these DNA:RNA hybrids (Marchena-Cruz et al. 2023). The ortholog of this in U. maydis was identified as UMAG_05214 and in S. cerevisiae as Rrp3 (Fig. 3, Table 1). We previously identified UMAG_05214 with upregulated transcript levels in the dormant teliospore, which remained at a steady state during germination (Seto et al. 2025). This suggests that UMAG_05214 may have an enhanced role during germination, and we hypothesize that it may function to regulate R-loop formation during transcription. UMAG_05214 may have an additional role in promoting the transcription of genes involved in maintaining cellular homeostasis in other cell types. Disruption in the function of this RNA helicase in U. maydis may cause irregular cell growth and dysregulation of cellular metabolism. Functional characterization is required to determine the impact of UMAG_05214 on teliospore germination and U. maydis growth. 24 IMA Fungus 16: e151785 (2025), DOI: 10.3897/imafungus.16.151785 Amanda M. Seto & Barry J. Saville: U. maydis RNA helicases UMAG_10683 was identified as the putative U. maydis Rok1/DDX52 ortholog (Fig. 2, Table 1). Our STRING analysis predicts that UMAG_10683 is involved in processing rRNA, suggesting that its function is conserved. In C. elegans, DDX-52 loss-of-function mutants were resistant to hypoxia, experienced arrested early development, and underwent cell death at high temperatures (Itani et al. 2021). In S. cerevisiae, Rok1 is an essential protein that functions to regulate cell cycle progression (Song et al. 1995; Giaever et al. 2002; Jeon and Kim 2010). S. cerevisiae cells with disrupted or overexpressed Rok1 are arrested at the G1/S phase of the cell cycle. This suggests that the protein levels of Rok1 regulate ribosome biogenesis at the beginning of the cell cycle (Jeon and Kim 2010). We hypothesize that deletion of UMAG_10683 will be lethal. If UMAG_10683 functions similarly to its orthologs, altering its expression will result in defects in cell cycle progression and altered growth rates. During U. maydis pathogenesis, the gene transcript increases and peaks at 2 dpi, indicating that enhanced transcription of UMAG_10683 may aid in the translation of genes during this stage of pathogenesis. In S. cerevisiae, RNA helicase Dbp3 null mutants were observed to have increased thermotolerance, oxidative stress resistance, DNA stress, and endoplasmic reticulum stress resistance (Delaney et al. 2013). The Arabidopsis thaliana orthologs are negative regulators of stress-responsive transcription activators (Kant et al. 2007; Khan et al. 2014). Overexpression studies of S. cerevisiae DBP3 suggested a role in protein secretion in fungal cells (Chen et al. 2023). Our phylogenetic and sequence analysis identified UMAG_01732 as the ortholog to Dbp3 in S. cerevisiae and STRS1 in A. thaliana. We did not identify orthologs in any other species included in our analysis (Fig. 3, Table 1). Our STRING analysis predicted interactions with proteins involved in ribosome biogenesis (Suppl. material 2: table S1), suggesting that this RNA helicase has a conserved function in fungi and plants. The UMAG_01732 gene transcript is upregulated in the dormant teliospore and decreases during teliospore germination (Seto et al. 2025). This transcript pattern suggests that this RNA helicase may be involved in the transition from a dormant state to one of high metabolic activity. We had named this RNA helicase udbp3 and created deletion mutants in the compatible haploid strains, 518 and 521 (Seto and Saville 2025). Deletion mutants were viable and showed no significant difference in dikaryon formation, pathogenesis, or teliospore formation and germination. We found that the deletion mutants were more tolerant to osmotic stress (Seto and Saville 2025). It was concluded that udbp3 may be a negative regulator of osmotic stress response by regulating a subset of stress-responsive genes during teliospore dormancy (Seto and Saville 2025). Future work should focus on creating overexpression mutants to determine if it functions similarly to DBP3. udbp3 may have a similar role in regulating the protein secretion pathway to ensure the development and growth of the promycelium during teliospore germination. Our STRING analysis for UMAG_03170 (Suppl. material 2: table S1) predicts that this putative RNA helicase functions during the synthesis of ribosomal subunits, suggesting a conserved role across eukaryotes. Its orthologs, Drs1 in S. cerevisiae and DDX27 in H. sapiens (Fig. 3, Table 1), have demonstrated roles during ribosome biogenesis (Table 5). The zebrafish ortholog, DDX27, contributes to the processing of ribosomal subunits and was also found to regulate the translation of genes involved in muscle growth (Bennett et al. 2018). During in planta infection, the gene transcript is found in the green-yellow module 25 IMA Fungus 16: e151785 (2025), DOI: 10.3897/imafungus.16.151785 Amanda M. Seto & Barry J. Saville: U. maydis RNA helicases (Table 2) (Lanver et al. 2018). Increased transcription of UMAG_03170 may indicate an increased requirement for ribosomes and may be involved in translating a subset of genes required for later stages of pathogenesis. UMAG_11281 is the putative ortholog to the protein Prp43 in S. cerevisiae. It is predicted to interact with proteins involved in splicing (Suppl. material 2: table S1). One notable interaction is with the U. maydis protein UMAG_01091, an rRNA processing protein called SAS10. The S. cerevisiae SAS10 ortholog has been shown to have a role in processing the 18S rRNA and has been linked to cell cycle progression (Bernstein and Baserga 2004). During U. maydis pathogenesis, the transcript is found in the green module, where the transcript level is low during early pathogenesis, increases, and remains upregulated during late pathogenesis (Table 2) (Lanver et al. 2018). This suggests an increased requirement for this RNA helicase during the later stages of pathogenesis. It may aid in cell cycle progression during in planta growth by modulating specific RNPs or facilitating protein-protein interactions during the synthesis of the 18S rRNA. The S. cerevisiae RNA helicase Dbp8 functions during the processing of the 40S ribosomal subunit. The U. maydis ortholog, UMAG_10241, is predicted to interact with proteins involved in rRNA processing (Suppl. material 2: table S1). One predicted interaction is with the U. maydis ortholog for Esf2, suggesting that UMAG_10241 may function similarly to the S. cerevisiae ortholog. Esf2 is an RNA-binding protein capable of enhancing the activity of Dbp8 and guiding it to its binding site (Granneman et al. 2006). The H. sapiens ortholog, DDX49, regulates the export of mRNAs and pre-ribosomal RNA levels, which results in the regulation of cell proliferation (Awasthi et al. 2018). The UMAG_10241 transcript is upregulated in the dormant teliospore compared to the haploid and dikaryon cell types and decreases during teliospore germination (Seto et al. 2025). The activity of this RNA helicase may be required to aid in the transition from dormancy to germination by promoting and regulating the translation of genes involved in cellular and metabolic activity. UMAG_05200 in U. maydis is the putative ortholog to the S. cerevisiae SF2 DEAD-box RNA helicase Dbp10 (Fig. 3, Table 1). STRING analysis predicts that UMAG_05200 interacts with proteins involved in ribosomal biogenesis (Suppl. material 2: table S1), suggesting that its function is conserved across eukaryotes. In S. cerevisiae, the helicase activity of Dbp10 is required to induce conformational changes to the pre-rRNA during the maturation of the 60S subunit (Burger et al. 2000; Mitterer et al. 2023). Knockdown and overexpression studies of the ortholog DDX54 in H. sapiens cell lines revealed that this RNA helicase responds to DNA damage by binding to pre-mRNAs involved in the DNA damage response pathway to increase their splicing rate and promote cell survival (Milek et al. 2017). The UMAG_05200 transcript is upregulated in the dormant teliospore, and the transcript level is maintained during teliospore germination (Seto et al. 2025). This upregulation during teliospore dormancy may indicate that the transcript is stored and required during germination to respond to potential DNA damage. DNA damage can be caused by genotoxic stressors such as UV exposure and ionizing irradiation, which can reduce fungal spore germination (reviewed in Braga et al. 2015). In the soilborne protozoan Spongospora subterranea, there is an enrichment of DNA repair genes during spore germination (Balotf et al. 2021). We hypothesize that UMAG_05200 functions during teliospore germination to protect the growing promycelium by regulating genes that respond to DNA damage. 32 IMA Fungus 16: e151785 (2025), DOI: 10.3897/imafungus.16.151785 Amanda M. Seto & Barry J. Saville: U. maydis RNA helicases RNA helicases with unknown functions Our sequence and phylogenetic analyses identified several RNA helicases that are either fungal-specific or whose function is unknown. We identified the SF1 Upf1-like RNA helicase UMAG_10130 (Figs 1, 6) and the SF2 DEAD-box RNA helicases UMAG_00835 and UMAG_05873 (Figs 3, 6) as putative basidiomycete-specific RNA helicases. The SF2 DEAH RNA helicase YLF419W in S. cerevisiae has orthologs only in the fungal species we assessed, and the U. maydis putative ortholog is UMAG_11114 (Figs 4, 6). The U. maydis gene UMAG_10130 is an SF1 Upf1-like RNA helicase, and the phylogenetic analysis shows a separate clade containing genes in the other basidiomycete fungi, S. reilianum, U. hordei, and C. neoformans, but not with any other species we assessed (Figs 2, 6). Sequence analysis indicates that UMAG_10130 contains sequence motifs typical of RNA helicases in the SF1 Upf1-like family, indicating that a helicase core is present. UMAG_10130 and its basidiomycete orthologs have not been characterized within their respective organisms. The STRING analysis (Suppl. material 2: table S1) predicts putative protein-protein interactions with the same proteins as UMAG_11428, the ortholog to Upf1. Upf1 is an RNA helicase that functions during RNA degradation, where it activates Figure 6. Maximum likelihood phylogenetic tree of RNA helicases with unknown function. The tree was constructed using orthologs of RNA helicases with unknown functions from U. maydis, S. reilianum, U. hordei, C. neoformans, S. cerevisiae, S. pombe, N. crassa, A. thaliana, C. elegans, D. discoideum, and H. sapiens, using W-IQ-TREE multicore version 1.6.12 with default settings (Trifinopoulos et al. 2016), 1000 ultrafast bootstrap replicates, and the approximate Bayes test. The tree was visualized with FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/), rooted with the SF1 clade as the outgroup. Bootstrap values are indicated at each node. Phylogenetic groups containing known RNA helicases were collapsed and labeled with their S. cerevisiae and H. sapiens orthologs. Clades highlighted in blue, red, green, and purple indicate RNA helicases with unknown functions. The scale bar represents the expected number of substitutions per amino acid. The full phylogenetic tree, including all gene names and species, is provided in Suppl. material 1: fig. S5. 2.0 sr12122 [Sporisorium reilianum] CNAG_04051 [Cryptococcus neoformans] Ucp12 [Schizosaccharomyces pombe] UMAG_05873 [Ustilago maydis] sr13565 [Sporisorium reilianum] UMAG_10130 [Ustilago maydis] UHO2_02332 [Ustilago hordei] YLR419W [Saccharomyces cerevisiae] UMAG_00835 [Ustilago maydis] sr14994 [Sporisorium reilianum] UHO2_00086 [Ustilago hordei] NCU01143 [Neurospora crassa] CNAG_05145 [Cryptococcus neoformans] Upf1/UPF1 Dhh1/DDX6 Rrp3/DDX47 sr16492 [Sporisorium reilianum] CNAG_00922 [Cryptococcus neoformans] CNAG_04022 [Cryptococcus neoformans] UHO2_07222 [Ustilago hordei] UHO2_00305 [Ustilago hordei] UMAG_11114 [Ustilago maydis] 94 96 100 92 85 100 100 100 87 100 96 100 100 82 100 94 100 100 81 100 100 100 100 100 100 100 100 SF 1 SF 2 DEAD-box DEAH/RHA DHX9 Dhr1/DHX37 33 IMA Fungus 16: e151785 (2025), DOI: 10.3897/imafungus.16.151785 Amanda M. Seto & Barry J. Saville: U. maydis RNA helicases the NMD pathway. UMAG_10130 may function similarly to UMAG_11428 in the NMD pathway. The gene transcript is found in all U. maydis cell types (Donaldson et al. 2017; Seto et al. 2025) and is found in the magenta module during pathogenesis (Table 2). The magenta module contains genes that have a significant role during the early stages of pathogenesis, more specifically during the establishment and maintenance of biotrophy (Lanver et al. 2018). The UMAG_10130 transcript is found in a different module from UMAG_11428, suggesting that it may assist in the degradation of a specific subset of genes during this stage of pathogenesis that is different from those targeted by UMAG_11428. The phylogenetic analysis identified two SF2 DEAD-box RNA helicases with no other closely related orthologs other than those found in the Basidiomycota. These RNA helicases have been identified as UMAG_05873 and UMAG_00835 (Figs 3, 6) and are uncharacterized proteins in U. maydis. Our findings suggest that these RNA helicases are basidiomycete-specific RNA helicases; however, more fungal species would need to be assessed for this interpretation to be conclusive. Sequence analysis revealed that both genes have a helicase core containing several canonical RNA helicase sequence motifs typical of this RNA helicase family (data not shown). The STRING analysis predicts that UMAG_05873 interacts with translational pathway proteins (Suppl. material 2: table S1), suggesting a role in translation. Analysis of the UMAG_05873 transcript during in planta infection places the transcript in the magenta module (Lanver et al. 2018). This module is correlated to biotrophic establishment and maintenance. The enhancement of this RNA helicase during this stage of pathogenesis suggests it may function to aid in the translation of transcripts required for changes in the growth of the mycelium during pathogenesis that may be specific to basidiomycetes. STRING analysis for UMAG_00835 predicts protein-protein interactions with ribosome biogenesis proteins, suggesting a role in processing and assembling ribosomes (Suppl. material 2: table S1). The UMAG_00835 transcript is present in the cyan/tumor module during in planta growth, which contains genes that respond to nutrient limitations on the plant surface (Table 2). The transcript is upregulated in the dormant teliospore and is decreased during germination (Seto et al. 2025). This transcript pattern suggests that UMAG_00835 is stored and is immediately translated during germination. Its presence in the Lanver et al. (2018) cyan/tumor module suggests that this RNA helicase may respond to changes in external nutrients. We hypothesize that when dormant teliospores are in a nutrient-rich environment, a decrease in this RNA helicase during germination promotes the translation of genes for cellular growth. An RHA-group SF2 DEAH RNA helicase YLF419W in S. cerevisiae was identified by Jankowsky (2011). Our analysis identified UMAG_11114 as the U. maydis ortholog. This RNA helicase is largely uncharacterized. Based on current research, it is a nonessential gene (Shiratori et al. 1999; Colley et al. 2000), and the protein has been found in both the mitochondria (Sickmann et al. 2003) and cytoplasm (Huh et al. 2003). YLF419W has protein sequence similarities to ancient eukaryotic retinoblastoma (Rb) protein, suggesting that YLF419W evolved from an ancient Rb gene, lost its Rb function, and instead gained the function as an RNA helicase (Takemura 2005). Our phylogenetic analysis (Figs 4, 6) shows that YLF419W clusters with putative RNA helicases in other fungal species. UMAG_11114 was identified as an RNA helicase that currently has an unknown function. The N. crassa ortholog was identified as msp-8 (NCU01143), and its putative function is in pre-mRNA splicing (Adhvaryu et al. 2016). Our 34 IMA Fungus 16: e151785 (2025), DOI: 10.3897/imafungus.16.151785 Amanda M. Seto & Barry J. Saville: U. maydis RNA helicases STRING analysis predicted protein-protein interactions with the same proteins as UMAG_00574, suggesting that this RNA helicase may have a redundant function. During U. maydis pathogenesis, the UMAG_11114 transcript is in the yellow module (Table 2). This transcript pattern indicates upregulation of the transcript during the mid to later stages of pathogenesis. This suggests a role in supporting post-transcriptional regulation of highly metabolic and cellular activity genes (Lanver et al. 2018). Further functional characterization in U. maydis is required to determine the role this RNA helicase has in RNA metabolism and if there is an impact on the progression of pathogenesis. Conclusion Annotating the RNA helicases of the basidiomycete U. maydis allowed the identification of their functions in relation to gene regulation and response to environmental factors. This provided insights into their potential roles during fungal growth and development. For pathogenic fungi, RNA helicases have the potential to modulate disease progression within their host. Our analysis identified 46 RNA helicases within U. maydis. Through a comprehensive review of the current research on their orthologs, we were able to make predictions of their functions and roles in the life cycle. We identified 28 RNA helicases that may contribute to U. maydis growth, stress response, pathogenesis, or teliospore dormancy and germination. Further characterization of these RNA helicases and their influence on the various aspects of the fungal life cycle and pathogenicity can aid in developing methods for mitigating or preventing fungal diseases. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Use of AI No use of AI was reported. Adherence to national and international regulations All the fungal strains used in this study have been legally obtained, respecting the Convention on Biological Diversity (Rio Convention). Funding Funding for this project was awarded by the Natural Sciences and Engineering Research Council (NSERC) of Canada to B.J.S. Author contributions The investigation, methodology for gathering and analyzing literature, formal analysis of data, visualization of data, and writing of the original draft were undertaken by A.M.S. Conceptualization of this review, supervision, funding acquisition, and the writing— review and editing—was provided by B.J.S. 35 IMA Fungus 16: e151785 (2025), DOI: 10.3897/imafungus.16.151785 Amanda M. Seto & Barry J. Saville: U. maydis RNA helicases Author ORCIDs Amanda M. 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Saville: U. maydis RNA helicases Supplementary material 1 Supplementary figures Authors: Amanda M. Seto, Barry J. Saville Data type: docx Explanation note: figure S1: The original maximum likelihood phylogenetic tree of SF1 Upf1-like RNA helicases; figure S2: The original maximum likelihood phylogenetic tree of SF2 DEADbox RNA helicases; figure S3: The original maximum likelihood phylogenetic tree of SF2 DEAH RNA helicases; figure S4: The original maximum likelihood phylogenetic tree of SF2 Ski2-lie RNA helicases. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/imafungus.16.151785.suppl1 Supplementary material 2 Supplementary tables Authors: Amanda M. Seto, Barry J. Saville Data type: xlsx Explanation note: table S1: STRING v. 12.0 – Predicted Functional Partners. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/imafungus.16.151785.suppl2