In the twenty years since the first genome sequencing of , the field has seen an explosion in both the number of sequenced genomes and our molecular understanding of this ubiquitous human fungal pathogen. Despite an improved knowledge of the genome, we still know little about the transcriptome, with key regulatory sequences like the untranslated regions of mRNA based only on in silico predictions and bulk-RNA-seq. Here, we provide an improved description of 5' and 3' untranslated regions of poly(A)-enriched RNA through experimental mapping of transcription start sites and polyadenylation sites using 5' and 3' End-Seq. We assigned high-quality 5' ends to 2,747 genes (average length 126 nt), 3' ends to 7,079 genes (average length 268 nt), and improved our understanding of the regulatory landscape of gene expression. We leveraged the refined 5' UTRs to identify upstream open reading frames and binding sites for important RNA binding proteins like the translational regulator Ssd1 and the 3' UTRs to define binding sites for PUF proteins known to contribute to mRNA localization and regulation. Although a single isoform typically dominated expression, we observed 148 instances of alternative start sites and 1,675 alternative stop sites. Interestingly, we detected multiple examples of premature transcriptional termination, including the first evidence for promoter-proximal premature transcriptional termination in a member of the Eurotiomycetes. Ultimately, we provide a resource to the community and an accurate starting point for unravelling the complexities of gene regulation in an important human pathogen.

RNA 2'-phosphotransferase Tpt1 is a widely distributed enzyme that removes an internal RNA 2'-phosphate by transfer to NAD+. Tpt1 is essential in fungi, where it erases the 2'-PO4 mark installed by tRNA ligase during tRNA splicing. Tpt1 executes a two-step reaction in which: (i) the RNA 2'-PO4 attacks NAD+ to form an RNA-2'-phospho-(ADP-ribose) intermediate and expel nicotinamide; and (ii) the ADP-ribose O2'' attacks the RNA 2'-phosphodiester to form 2'-OH RNA and ADP-ribose-1'',2''-cyclic phosphate products. All Tpt1 enzymes studied to date are monofunctional units comprising a single bilobed fold composed of an RNA-binding lobe and an NAD+-binding lobe. We now find that fission yeast Tpt1 is an exception to this rule. Schizosaccharomyces pombe Tpt1 (SpTpt1) consists of an N-terminal RNA 2'-phosphotransferase catalytic domain (aa 1-237) linked to a C-terminal domain (aa 238-365) homologous to budding yeast iron-sulfur cluster assembly factor Yae1. The SpTpt1 catalytic domain and the Yae1 domain are both essential for S. pombe growth, though they need not be linked within the same polypeptide. A mutational analysis of the 2'-phosphotransferase domain illuminates the distinct contributions of essential active site constituents Arg50 and Arg96 during the two chemical steps of the Tpt1 pathway.

Small RNAs play essential roles in gene regulation across diverse biological processes. Crosslinking, ligation, and sequencing of hybrids (CLASH) experiments have revealed that PIWI and Argonaute proteins can each bind a wide range of mRNA targets with distinct base-pairing rules, raising questions about the flexibility and functional relevance of these interactions. Given that crosslinking-induced mutations (CIMs) provide single-nucleotide resolution molecular footprints of RNA-binding proteins, we developed MUTACLASH, a bioinformatics tool for systematically analyzing CIMs in CLASH datasets. Our analyses indicate that CIMs function as molecular footprints of Argonaute binding on target mRNAs. Specifically, for C. elegans miRNA and piRNA CLASH data, CIMs are enriched at the center of small RNA binding sites, as well as at nucleotides within mRNA target sites that exhibit local mismatches in piRNA interactions. Furthermore, we show that mRNAs with non-canonical miRNA and piRNA binding sites and/or low hybrid abundance marked by CIMs exhibit stronger regulatory effects than those without CIMs, demonstrating the utility of CIM analysis in identifying functional small RNA binding sites, including those that are otherwise likely overlooked with current analysis tools.

Eukaryotic translation initiation is critically regulated by 5' UTR features, including uORFs, Kozak sequences, and secondary structures, that modulate ribosome dynamics. Although canonical mRNAs dominate protein synthesis, ribosome profiling and peptidomics reveal ribosomes actively engaging putative non-coding RNAs (ncRNAs), translating enigmatic short ORFs (sORFs). We systematically analyzed 5' UTR architectures across canonical mRNAs, ribosome-associated ncRNAs (translationally active), and non-translated ncRNAs using curated human datasets. mRNAs exhibited optimal translational features (short 5' UTRs, few uORFs), while translated ncRNAs showed intermediate features, and non-translated ncRNAs the weakest. Notably, mRNAs with long 5' UTRs maintained high translational efficiency through conserved regulatory elements. Integrating these features into our newly developed random forest model, plusCE, surpassed existing methods in predicting translation efficiency, suggesting their potential relevance to translation mechanisms and providing guidance for rational 5' UTR design to modulate translation. Although some ncRNAs are frequently bound by ribosomes, they show no evidence of stable translation, consistent with their lack of coding-related evolutionary signatures. Our analysis suggests that ribosome-bound ncRNAs may not reflect adaptive evolution toward coding function, but rather represent a reservoir of untranslated transcripts that engage the translation machinery through permissive sequence features. Together, these results demonstrate that ribosome engagement is primarily shaped by 5' UTR sequence features, highlighting the importance of regulatory grammar in translation control and complementing current models of ncRNA evolution.

Although protein-RNA interactions are crucial for many biological processes, predicting their binding free energies (ΔG) is a challenging task due to limited available experimental data and the complexity of these interactions. To address this issue, we developed a machine learning-based model designed to predict energy-based scores for protein-RNA complexes, called PANTHER score. By applying a local-to-global approach, the here proposed methodology can be subdivided into four steps: (1) we derived 87,117 pairwise local interaction energies out of 331,744 obtained from molecular dynamics simulations for a training set composed by 46 curated protein-RNA complexes; (2) we trained ML models derived from pairwise interaction features to predict the local interaction energies without performing MD runs; (3) we integrated the predicted local interaction energies with our here proposed local-to-global methodology, to calculate the model-specific PANTHER score; (4) we test the model-specific PANTHER score on a test set of 7 complexes (5) we further exposed all the models to an external stress set which includes 110 complexes with experimental ΔG allowing for final selection of the optimal model for implementation in the PANTHER scoring pipeline. Among all the multiple regression models developed here and evaluated on the test set, Random Forest Regression exhibited the highest predictive performance as a model-specific PANTHER score, with a Pearson correlation coefficient of (r) of 0.80 and mean absolute error (MAE) of 1.79 kcal/mol. Furthermore, the Random Forest Regression model maintained strong predictive capabilities on the stress set as well with (r) of 0.64 and MAE of 1.63 kcal/mol. Benchmarking against existing tools on the stress test set, the PANTHER score demonstrated superior accuracy and reliability. This study highlights the effectiveness of machine learning in addressing data limitations through innovative strategies, positioning here proposed PANTHER score as a valuable tool for predicting protein-RNA binding affinities in biomolecular research and drug discovery.

Nicotinamide adenine dinucleotide (NAD) is a ubiquitous enzyme cofactor that serves as a carrier of hydride ions for metabolic oxidation-reduction reactions. NAD is also sometimes used as a source of activated adenosine monophosphate (AMP) for adenylation reactions or as a precursor of ADP-ribose upon removal of nicotinamide. Many bacterial riboswitch classes are known to sense nucleotide-derived enzyme cofactors, but NAD is one of several ancient cofactors that have few or no known riboswitch representatives. Two rare riboswitch classes, named NAD+-I and NAD+-II, have been reported that regulate genes relevant to NAD biosynthesis and transport. However, these RNAs exhibit unusual functional and structural properties. Here we report that miniature NAD+-II riboswitches, named mini-NAD+-II, are more abundant and widespread than the longer RNAs that were used to defined the original consensus model for this class. The newfound examples are commonly found within lactic acid bacteria, which are notable for varied metabolic fermentation strategies used to maintain sufficient NAD+. Furthermore, the simple H-type pseudoknot core of mini-NAD+-II aptamers is similar to that of class I preQ1 riboswitch (preQ1-I) aptamers. Thus, H-type pseudoknots might serve as a versatile architecture for the natural or synthetic construction of ligand-binding aptamers.

The 5'-N7-methylated guanosine triphosphate cap structure plays a critical role in mRNA translation and mRNA stability. The recent invention of co-transcriptional capping of mRNAs using Trinucleotide Capped Primers (TCP) allowed for development of large-scale in vitro transcription (IVT) synthesis of mRNA carrying a eukaryotic Cap 1 structure (TCP-mRNA). Here we present a novel "one-pot-two-step" methodology for the synthesis of TCPs that improves the yield and simplifies the isolation and purification of the TCPs. Over 70 different modified TCPs, the analogs of 7mGpppAmpG trimer, were synthesized, characterized, and tested for their ability to initiate IVT reaction. Results demonstrate that full complementarity of TCP to a template strand of dsDNA template at transcription initiation (start) site, at positions +1 and +2, is required and sufficient to obtain capped TCP-mRNA with high capping efficiency (>98%) and high yield (>5 mg/mL). The developed approach can be applied for small- and large-scale mRNA synthesis carrying various 5'-cap structures.

Magnesium is vital for bacterial survival, and its homeostasis is tightly regulated. Intracellular pathogens like (Mtb) often face host-mediated magnesium limitation, which can be counteracted by upregulating the expression of Mg²⁺ transporters. This upregulation may be via Mg²⁺-sensing regulatory RNA such as the Mbox riboswitch, which acts as a transcriptional "OFF-switch" under high Mg²⁺ conditions. Mtb encodes two Mbox elements with strong similarity to the Mbox. In the current study, we characterize the Mbox encoded upstream of the Mtb operon, which is required for growth in low Mg²⁺/low pH. We show that this switch operates via a translational expression platform and Rho-dependent transcription termination, which is the first such case reported for an Mbox. Moreover, we show that the switch directly controls a small ORF encoded upstream of We have annotated this highly conserved uORF , but its role remains unclear. Interestingly, a homologous gene exists outside the Mbox-regulated context, suggesting functional importance beyond magnesium stress. Overall, this study uncovers a dual mechanism of riboswitch-regulation in Mtb, combining translational control with Rho-mediated transcription termination. These findings expand our understanding of RNA-based gene regulation in mycobacteria, with implications for pathogenesis and stress adaptation.

Hepatitis C virus (HCV) is a major global health burden, associated with chronic liver diseases including cirrhosis and hepatocellular carcinoma. Viral replication critically depends on conserved cis-acting RNA elements (CREs), such as the 5BSL3.2 stem-loop near the 3' end of the open reading frame. This element forms a long-range kissing-loop interaction with the SL2 domain of the 3'X tail, essential for efficient genome replication. However, the role of host RNA-binding proteins (RBPs) in regulating this RNA-RNA interaction remains poorly understood. To explore this, we investigated whether the host RBP hnRNPA1 modulates HCV replication by targeting the 5BSL3.2 element. Using an integrated approach combining structural biology, biophysics, and biochemical assays, we identify the terminal loop of 5BSL3.2 as a high-affinity binding site for the tandem RNA recognition motifs (RRMs) of hnRNPA1. Our data reveal that adenine-rich residues within the loop are critical for binding specificity. Our results uncover a structural mechanism by which hnRNPA1 binding perturbs the kissing-loop interaction between 5BSL3.2 and the SL2 element of the viral 3'X-tail, which impacts viral replication. This study highlights a previously unrecognized role of hnRNPA1 in modulating viral RNA structure and suggests a novel interface for host-directed antiviral intervention.

The transcription of human telomeres gives rise to a family of long noncoding RNAs, named TERRA. We previously showed that TERRA transcription is driven by CpG island promoters that are composed by stretches of three types of repeats. Using the human genome assembly that was available at that time, putative promoter sequences were localized at several subtelomeres. In this work, using the T2T-CHM13v2.0 human reference genome, we found that 39 out of 46 subtelomeres contain TERRA promoters and grouped them in classes depending on their organization. We then discovered 106 intrachromosomal TERRA-like promoters, adjacent to interstitial telomeric sequences (ITSs) or far away from them. Fortyseven of these promoters are flanked and may regulate the transcription of coding genes, ncRNAs or pseudogenes. Comparative sequence analysis showed that interstitial and subtelomeric promoters belong to a previously undescribed family of segmental duplications deriving from common ancestral sequences. RT-PCR experiments in seven cell lines demonstrated that TERRA transcripts can be synthesized from ITSs. TERRA expression was always low in primary fibroblasts and HeLa cells while highly variable in the other two telomerase positive (HT1080 and HEK293) and in the three telomerase negative ALT cell lines (GM847, U2OS and VA13). The analysis of RNA-seq data from U2OS, HeLa and HEK293 cells showed that 205 ITSs were transcribed in at least one cell lines. The fraction of transcribed ITSs and the level of their transcription increased with the length of the telomeric repeat stretch. Given the large number of transcribed ITSs, we propose that these loci contribute significantly to the production of the TERRA pool.

The RNase II/RNB family of exoribonucleases is present in all domains of life and includes three main eukaryotic members, the Dis3-like proteins (Dis3, Dis3L1, and Dis3L2). At the cellular level, Dis3L2 is distinguished by the unique preference for uridylated RNA substrates and the highest efficiency in degrading double-stranded RNA. Defects in these enzymes have been linked to some types of cancers and overgrowth disorders in humans. In this work, we used the Dis3L2 protein from the model organism (SpDis3L2) to better understand the mechanism of action of Dis3-like exoribonucleases, and to elucidate how single amino acid substitutions in these proteins can affect the biochemical properties of the enzymes, potentially contributing to the molecular basis of the related human diseases. We determined the crystal structure of SpDis3L2 bound to a U RNA, in which the protein displays a typical vase-like conformation, accommodating 6 nucleotides of the RNA 3'-end. Furthermore, we constructed two SpDis3L2 protein variants, harboring single amino acid substitutions mimicking the ones already found in human patients, to test their catalytic activity in vitro. We highlight the A756R SpDis3L2 variant, which loses the ability to degrade double-stranded RNA substrates and accumulates intermediate degradation products when degrading single-stranded RNA substrates. As such, A756 seems to be a key residue responsible for the normal cellular function of Dis3L2, specifically regarding its important role in the degradation of structured RNA substrates.

Riboswitches are noncoding mRNA regions that regulate gene expression by sensing small molecules. While most riboswitches turn off gene expression, guanidine-I family riboswitches enhance gene expression. Although crystal structures provide insights into the structural basis for guanidinium ion (Gdm) sensing by the guanidine-I riboswitch aptamer (GRA) domain, the mechanistic interplay between ligand and metal ion binding, RNA conformational changes, and regulatory function remains poorly understood. Using molecular dynamics simulations, we explore the combined effects of the positively charged Gdm, Mg, and K observed in close proximity in the experimental crystal structure on the GRA structural dynamics. Our simulations reveal that the binding pocket frequently transitions between ligand bound-like and unbound-like states in the absence of divalent ions, while Mg stabilizes a bound-like RNA conformation. Furthermore, both Mg and Gdm facilitate K positioning near the binding pocket. As a result, Mg, Gdm, and K synergistically increase the structural rigidity of the GRA domain, particularly the P2-P3 junction and the 3' end near the terminator stem. This enhances localized interactions that pull the P1a and P3 domains together to make the transcriptional control region available for expression. Our proposed mechanism is fully consistent with experimental structural and biochemical (including isothermal titration calorimetry and structure-guided mutagenesis) data and rationalizes how the unique triad of ions works together to influence the conformational dynamics of the aptamer domain and riboswitch function. This information can guide future synthetic riboswitch design and the identification of novel therapeutic targets beyond static structural information.

Numerous RNA modifications are known in prokaryotes, but their dynamics and function in regulation remain largely unexplored. In , three methyltransferases catalyze the 5-methylcytosine (mC) modification in ribosomal RNA. Here, we introduce mC-Rol-LAMP (mC-rolling circle loop-mediated isothermal amplification) as a novel qPCR-based method that offers high sensitivity and site-specific resolution to detect and quantify mC in total RNA. When applying mC-Rol-LAMP to under heat stress (45 °C), we observe a site-specific increase of mC at position 1407 of 16S rRNA from 77% to 89%, while mC levels at positions 967 (16S) and 1962 (23S) remain unchanged. In recovered cells (at 37 °C), the mC abundance partially returns to the no-stress level. Under oxidative stress, the level of mC1407 also increases, but remains high in recovered cells. These results demonstrate for the first time a reversible, stress-dependent, and site-specific change in the rRNA modification level of a bacterium. mC-Rol-LAMP is a powerful and easy-to-use tool for studying mC in all RNA species, allowing the quantitative and site-specific detection of this modification.

The ATP-dependent RNA helicase Up-frameshift 1 (UPF1) is an essential protein in mammalian cells and a key factor in nonsense-mediated mRNA decay (NMD), a translation-dependent mRNA surveillance process. UPF1 is mainly cytoplasmic at steady state but accumulates in the nucleus after inhibiting CRM1-mediated nuclear export by leptomycin B (LMB), indicating that UPF1 shuttles between the nucleus and the cytoplasm. Consistent with its dual localization, there is evidence for nuclear functions of UPF1, for instance, in DNA replication, DNA damage response, and telomere maintenance. However, whether any of UPF1's biochemical activities are required for its nuclear-cytoplasmic shuttling remains unclear. To investigate this, we examined two UPF1 mutants: the well-described ATPase-deficient UPF1-DE (D636A/E637A) and a newly generated RNA-binding mutant UPF1-NKR (N524A/K547A/R843A). Biochemical assays confirmed that the UPF1-NKR mutant cannot bind RNA or hydrolyze ATP in vitro but retains interaction with UPF2, UPF3B, and SMG6. Overexpression of UPF1-NKR exerted a dominant-negative effect on endogenous UPF1 and inhibited NMD. Subcellular localization studies revealed that UPF1-DE accumulates in cytoplasmic granules (P-bodies), even in the presence of LMB, whereas UPF1-NKR shuttles normally. This indicates that UPF1's shuttling does not require its RNA-binding or ATPase activities. Notably, the UPF1-DE.NKR double mutant restored nuclear-cytoplasmic shuttling and prevented accumulation in P-bodies, suggesting that the shuttling defect of UPF1-DE arises from its tight binding to RNA. Overall, our findings demonstrate that UPF1's shuttling is independent of its ATPase and RNA-binding activities, with RNA binding itself being a key determinant of its cytoplasmic retention.

Gene therapy using recombinant adeno-associated viral (AAV) vectors is a promising approach for treating a wide range of inherited diseases. Precise characterization of the AAV vector genomes and their transcripts is essential for further optimization of this technique. Current visualization methods require multiple assays for detecting DNA and RNA, often involving mRNA-to-cDNA conversion. This can obscure insights into spatial distributions, particularly when AAV DNA and mRNA exhibit divergent trends. To address this challenge, we developed a novel padlock probe (PLP)-based rolling-circle amplification (RCA) technique. Using SplintR DNA ligase, which ligates single-stranded DNA splinted by complementary RNA sequences, enabled our method to directly target AAV mRNA without requiring conversion to cDNA, as well as genomic DNA at the same time. Furthermore, incorporating an intron into the transgene sequence allowed for the application of specific probes that differentiate between transgene DNA and its mRNA transcripts. This method can simultaneously and specifically detect AAV transgene DNA and its mRNA, with probes targeting AAV single-stranded DNA (+), AAV single-stranded DNA (-), and mRNA effectively amplifying each target. Furthermore, this approach enables to differentiate AAV single-stranded from double-stranded DNA by a combined treatment of Lambda Exonuclease and restriction enzyme digestion, providing the possibility for tracking of AAV genome process following transduction. In transduced HeLa cells and liver tissues harvested from AAV-injected mice, using PLP-RCA we were able to observe distinct temporal patterns in AAV DNA and mRNA distributions over time, revealing early instability in AAV DNA that impacted transduction efficiency. This novel approach provides a robust tool for in situ analysis of AAV genome and transcript distributions at the single-cell level.

RNA-binding proteins (RBPs) and microRNAs (miRNAs) play crucial roles in regulating gene expression at the post-transcriptional level in tumorigenesis. They primarily target the 3'UTRs of mRNAs to control their translation and stability. However, their co-regulatory effects on specific mRNAs in the pathogenesis of particular cancers are yet to be fully explored. CSDE1 is an RBP that promotes melanoma metastasis, and the mechanisms underlying its function in melanoma development are yet to be fully understood. Here, we report that CSDE1 enhances TMBIM6 protein expression without altering its mRNA levels in melanoma cells, indicating post-transcriptional regulation. CSDE1 and AGO2 competitively bind to TMBIM6 mRNA, and we identify miR-20-5p, which represses TMBIM6 expression, regulates the binding of CSDE1 to TMBIM6 mRNA. Further, the RNA-binding mutant of CSDE1 showed reduced affinity towards TMBIM6 mRNA, thus allowing AGO2-mediated silencing of TMBIM6 expression. Our study highlights the pivotal role of CSDE1 in regulating miR-20a-5p function and the expression of TMBIM6 in melanoma cells, thus unveiling the potential of therapeutic strategies targeting this regulatory pathway in treating malignant skin cancers.

Proteins have traditionally been understood through their tertiary structures, with well-defined conformations considered essential for biological function. This classical structure-function paradigm implies that proteins with high intrinsic disorder would be less critical for cellular survival. Recent discoveries have suggested that some intrinsically disordered proteins or even fully disordered proteins without any apparent tertiary structures are essential. However, the biological significance of such disordered proteins is not comprehensively understood. Here, using genome-wide CRISPR screening, we demonstrated that highly or fully disordered proteins show comparable essentiality to well-folded proteins. We found that the proportion of essential proteins is comparable across proteins of varying disorder levels, although structured proteins are more prevalent among essential genes. Focusing on FAM32A, one of the essential, fully disordered proteins identified in our screen, we show that its depletion leads to increased intron retention and downregulation of many other essential genes. These findings reshape our understanding of the structure-function paradigm, highlighting that fully disordered proteins can be essential for cellular viability.

Maturation of protein-coding precursor messenger RNA (pre-mRNA) is closely linked to RNA polymerase II (Pol II) transcription. However, the mechanistic understanding of how RNA processing is coordinated with transcription remains incomplete. Conserved proteins interacting with the C-terminal domain of the largest catalytic subunits of Pol II and nascent RNA (CID-RRM factors) were demonstrated to play a role in pre-mRNA 3'-end processing and termination of Pol II transcription. Here, we employ a fully reconstituted system to demonstrate that the fission yeast CID-RRM factor Seb1 acts as a bona fide elongation factor in vitro. Our analyses show that Seb1 exhibits context-dependent regulation of Pol II pausing, capable of either promoting or inhibiting pause site entry. We propose that CID-RRM factors coordinate Pol II transcription and pre-mRNA 3'-end processing by modulating the rate of Pol II transcription.

Alternative splicing (AS) enables cells to produce multiple protein isoforms from single genes, fine-tuning protein function across numerous cellular processes. However, despite its biological importance, researchers lack effective tools to compare the domain composition of AS-derived protein isoforms because such comparisons require both structural data and specialized methods. Recent advances in AI-driven protein structure prediction, particularly AlphaFold2, now make accurate structural determination of splicing isoforms accessible, enabling functional AS analysis at the protein structure level. Here, we present IsoformMapper, a web resource that analyzes AS through network community analysis of protein structures. This approach captures 3D physical interactions between protein regions often missed by traditional domain analysis, enabling structural comparisons of isoforms across any biological system. We illustrate our tool by analyzing validated human Bcl-X protein isoforms, revealing how AS creates distinct community structures with antagonistic functional roles. As a proof of concept, we apply our tool to investigate how GENOMES UNCOUPLED1 (GUN1)-dependent retrograde signaling regulates plant de-etiolation through alternative splicing in Arabidopsis. In response to light, gun1 shows alterations in spliceosome component expression, suggesting that GUN1 contributes to AS regulation of genes essential for photosynthetic establishment. The gun1 mutant displays altered splice variant ratios for PNSL2, CHAOS, and SIG5. Our tool reveals that these isoforms form distinct protein community structures, demonstrating how AS impacts protein function and validating IsoformMapper's practical value.

Precise recognition of the boundaries between exons and introns (splice sites, SS) is essential for the fidelity of gene expression. In contrast with the 5'SS, the consensus 3'SS sequence in both and humans is just three nucleotides long: YAG. How the correct 3'SS is chosen among many possible alternates by the spliceosome is often unclear but likely involves proofreading by the Prp22 ATPase. In cryo-EM structures of spliceosome product (P) complexes, glutamine 1594 in the highly conserved α-finger domain of the Prp8 protein interacts directly with the -3 pyrimidine of the 3'SS. To investigate the role of this interaction, we constructed a Prp8 mutant and studied the impact on splicing and 3'SS selection. Using splicing reporter assays and RNA-seq, we show that Prp8 enables use of non-consensus 3'SS by relaxing sequence requirements at the -3 and -2 positions. Consequently, this can change how adjacent 3'SS compete with one another during mRNA formation. The ability for Prp8 to support splicing at non-YAG sites depends on the splicing factors Prp18 and Fyv6, and Prp8 has genetic interactions with Prp22 mutants. Together, these findings suggest that the Prp8 α-finger acts as a sensor of 3'SS accommodation within the spliceosome active site. We propose that conformational change of the α-finger either allows or inhibits binding of the Prp22 c-terminal tail. This may provide a mechanism for regulating Prp22 activity in response to 3'SS binding.