U1 snRNA Research Articles

Examining the capacity of human U1 snRNA variants to facilitate pre-mRNA splicing.

The human U1 snRNA is encoded by a multigene family consisting of transcribed variants and defective pseudogenes. Many variant U1 (vU1) snRNAs have been demonstrated to not only be transcribed but also processed by the addition of a trimethylated guanosine cap, packaged into snRNPs, and assembled into spliceosomes; however, their capacity to facilitate pre-mRNA splicing has, so far, not been tested. A recent systematic analysis of the human snRNA genes identified 178 U1 snRNA genes that are present in the genome as either tandem arrays or single genes on multiple chromosomes. Of these, 15 were found to be expressed in human tissues and cell lines, although at significantly low levels from their endogenous loci, <0.001% of the canonical U1 snRNA. In this study, we found that placing the variants in the context of the regulatory elements of the RNU1-1 gene improves the expression of many variants to levels comparable to the canonical U1 snRNA. Application of a previously established HeLa cell-based minigene reporter assay to examine the capacity of the vU1 snRNAs to support pre-mRNA splicing revealed that even though the exogenously expressed variant snRNAs were enriched in the nucleus, only a few had a measurable effect on splicing.

Drosophila Amus and Bin3 methylases functionally replace mammalian MePCE for capping and the stabilization of U6 and 7SK snRNAs.

U6 and 7SK snRNAs have a 5' cap, believed to be essential for their stability and maintained by mammalian MePCE or Drosophila Bin3 enzymes. Although both proteins are required for 7SK stability, loss of neither destabilizes U6, casting doubts on the function of capping U6. Here, we show that the Drosophila Amus protein, homologous to both proteins, is essential for U6 but not 7SK stability. The loss of U6 is rescued by the expression of an Amus-MePCE hybrid protein harboring the methyltransferase domain from MePCE, highlighting the conserved function of the two proteins as the U6 capping enzyme. Our investigations in human cells establish a dependence of both U6 and 7SK stability on MePCE, resolving a long-standing uncertainty. While uncovering a division of labor of Bin3/MePCE/Amus proteins, we found a "Bin3-Box" domain present only in enzymes associated with 7SK regulation. Targeted mutagenesis confirms its importance for Bin3 function, revealing a possible conserved element in 7SK but not U6 biology.

G3BP1 and SLU7 Jointly Promote Immune Evasion by Downregulating MHC-I via PI3K/Akt Activation in Bladder Cancer.

Immune checkpoint inhibitors (ICIs) show promise as second-line treatment for advanced bladder cancer (BLCA); however, their responsiveness is limited by the immune evasion mechanisms in tumor cells. This study conduct a Cox regression analysis to screen mRNA-binding proteins and reveals an association between Ras GTPase-activating protein-binding protein 1 (G3BP1) and diminished effectiveness of ICI therapy in patients with advanced BLCA. Subsequent investigation demonstrates that G3BP1 enhances immune evasion in BLCA cells by downregulating major histocompatibility complex class I (MHC-I) through phosphoinositide 3-kinase (PI3K)/Akt signaling activation. Mechanistically, G3BP1 interacts with splicing factor synergistic lethal with U5 snRNA 7 (SLU7) to form a complex with poly(A)-binding protein cytoplasmic 1 and eukaryotic translation initiation factor 4 gamma 1. This complex stabilizes the closed-loop structure of the mRNAs of class IA PI3Ks and consequently facilitates their translation and stabilization, thereby activating PI3K/Akt signaling to downregulate MHC-I. Consistently, targeting G3BP1 with epigallocatechin gallate (EGCG) impedes immune evasion and sensitizes BLCA cells to anti-programmed cell death (PD)-1 antibodies in mice. Thus, G3BP1 and SLU7 collaboratively contribute to immune evasion in BLCA, indicating that EGCG is a precision therapeutic agent to enhance the effectiveness of anti-PD-1 therapy.

RNA Splicing Factor Mutations Drive Aberrant Canonical and Cryptic Circular RNA Biogenesis in Leukemia

RNA splicing factors (SFs) are recurrently mutated in liquid cancers. Mutations on SFs such as SF3B1, SRSF2, U2AF1, ZRSR2, and the U1 snRNA promote leukemogenesis by driving aberrant pre-mRNA splicing in critical pathways such as DNA damage response (DDR) and cell cycle regulation (CCR). Circular RNAs (circRNAs) are covalently closed RNAs that form via backsplicing ( Fig A). As a novel RNA species, circRNAs are now known to play diverse functional roles in cancer biology. Because backsplicing is a type of alternative splicing, we hypothesized that SF mutations also influence circRNA biogenesis in leukemia. To date, such causal links have not been clearly established. We first performed circRNA-seq on a panel of lymphoid and myeloid cell lines modeling the most common SF mutations ( SF3B1 K700E, SRSF2 P95H, U2AF1 S34F/Q157R, ZRSR2 KO, and U1 snRNA 3A&amp;gt;C). We observed that SF mutations tend to globally upregulate circRNAs with each mutant SF driving its own mutually exclusive set of circRNAs that we have termed SF mutation-associated circRNAs (SF-circRNAs). Each set of SF-circRNAs differs from each other along several features such as average lengths and selection of backsplicing exons. Additionally, while SF-circRNAs were generally mutation-specific, the majority of SF-circRNAs arose from commonly shared genes enriched for the DDR and CCR pathways. These results support a model in which SF mutations drive divergent splicing events convergent on a few pathways as a common mechanism in leukemias. Because SF3B1 is the most recurrently mutated SF in cancers, we concentrated on studying SF3B1 mutation-associated circRNAs (SF3B1-circRNAs). During linear RNA splicing, hotspot mutations on SF3B1 typically promote selection of cryptic 3' splice site (SS). Cryptic 3'SS are weak, inauthentic 3'SS proximal to the canonical site that can be activated for splicing by mutant SF3B1. Reasoning that cryptic 3'SS selection also occurs during backsplicing, we developed a pipeline called CrypticCIRC to call for these events. Through CrypticCIRC, we discovered the existence of cryptic circRNAs that account for nearly 1 in 10 of all SF3B1-circRNAs. Approximately 25% of cryptic circRNAs emanated from de novo 3'SS previously unannotated in the genome. Furthermore, we found that cryptic circRNAs were especially enriched on genes involved in the DDR and CCR pathways, furthering the notion that these pathways are preferentially targeted by SF mutations. Finally, by comparing with linear RNA splicing data, we also observed that some cryptic 3'SS may undergo either aberrant linear splicing or aberrant backsplicing but not both, indicating mutual exclusivity in cryptic splice site selection between linear and backsplicing. Finally, to mechanistically dissect SF-circRNA biology, we focused our study on one candidate circRNA, circZEB1. The parental ZEB1 gene, a master transcription and DDR factor, undergoes both aberrant canonical and cryptic backsplicing at exon 2 with exon 4 to give rise to two circZEB1 isoforms ( Fig B). In our full SF mutant panel, we found that the canonical and cryptic isoforms were consistently upregulated only in SF3B1 mutant cell lines (Nalm-6, K-562, HEK293T, HG-3, and MEC-1); We further confirmed circZeb1 upregulation in mouse models (CD19-Cre Sf3b1 K700E) where the canonical isoform is evolutionarily conserved. These results confirm circZEB1 as a true bona fide SF3B1-circRNA. Focusing on the function of this circRNA, we found that CasRx-mediated knockdown of canonical circZEB1 resulted in increased ZEB1 protein, but not mRNA, expression while overexpression of circZEB1 decreased ZEB1 protein levels. These results show that circZEB1 translationally regulates its parental gene. Finally, consistent with ZEB1's role in genome stability, we also observed a global reduction in γH2AX levels concomitant with circZEB1 ablation, highlighting DDR as a common pathway for SF-circRNAs ( Fig B). Altogether, we demonstrate that SF mutations drive widespread aberrant circRNA biogenesis mainly on DDR and CCR genes. We provide evidence for the existence of cryptic circRNAs as a novel class of circRNAs. We show that circZEB1 promotes DNA damage via regulating ZEB1 translation. Our results collectively highlight circRNAs as an additional regulatory modality alongside linear RNAs through which SF mutations exert function, supporting a unifying mechanism for the pan-cancer impact of SF mutations in leukemia.

Dysregulated Alternative Splicing in Inv(16) Acute Myeloid Leukemia Via U2AF1 Deacetylation during DNA Damage Response

Inversion of chromosome 16, inv(16) [inv(16)(p13q22) or t(16;16)(p13.1;q22)], is a recurrent chromosomal translocation observed in 5-8% of acute myeloid leukemia (AML) cases. Inv(16) creates a fusion gene CBFb-MYH11 (CM) which impairs hematopoietic differentiation and creates abnormal progenitor populations prone to leukemic transformation. We reported that CM interacts with HDAC8 and enhances its activity, and high HDAC8 activity impaired DNA damage response (DDR) in CM knock-in (KI) hematopoietic stem and progenitor cells (HSPCs) (Zhang, L et al, ASH meeting 2022, https://doi.org/10.1182/blood-2022-160280). The recruitment of BRCA1-mRNA splicing machinery is critical for efficient DDR and the U2 small nuclear RNA auxiliary factor 1 (U2AF1) is required for accurate 3'-splice site selection. Here, we show how U2AF1 is post-translationally regulated following DNA damage and its impact on alternative splicing in CM KI HSPCs upon DNA damage. First, we identified TIP60 as the histone acetyltransferase (HAT) responsible for the increased U2AF1 acetylation induced by ionizing radiation (IR). We detected enhanced interaction between U2AF1 and TIP60 but not with other HATs (GCN5, PCAF, CBP) along with the increased U2AF1 acetylation in response to IR. In addition, TIP60 knockdown (KD) reduced U2AF1 acetylation, while TIP60 expression increased U2AF1 acetylation, indicating that TIP60 mediates the acetylation of U2AF1. To pinpoint the specific lysine residue subjected to TIP60 catalysis, we introduced lysine site-specific mutants U2AF1-HA-K15R, K23R, K39R, K175R, or 4 lysine sites mutant (U2AF1-HA-4mut) with TIP60. Co-immunoprecipitation (anti-HA) followed by western blotting (anti-acetyl-lysine) showed undetectable acetylation of U2AF1-HA-K23R and K175R mutants (similar to U2AF1-HA-4mut), suggesting that K23 and K175 residues are essential for TIP60-mediated acetylation. To investigate the impact of K23 or K175 acetylation on the assembly of the BRCA1-mRNA splicing complex and DDR, we subjected U2AF1-K23R or K175R expressing 32D cells to IR (3.5 Gy). Both U2AF1-K23R and K175R mutants exhibited markedly reduced acetylation levels after IR, confirming that K23 and K175 sites are the acetylation sites responding to DNA damage. Furthermore, the interaction of U2AF1-K23R and K175R with BRCA1 and BCLAF1 was impaired. We observed increased apoptosis in U2AF1-K23R and K175R cells compared to wild-type (WT) U2AF1 cells (K23R vs WT, 51.975±7.867% vs 6.055±0.634%, P=0.0011; K175R vs WT,22.645±3.037% vs 6.055±0.634%, P=0.0017) after KD of endogenous U2AF1 and subjected to IR (3.5 Gy). These results indicate that U2AF1 is acetylated by TIP60 upon DNA damage on K23 and K175 which is critical for cell survival and assembly of the BRCA1-mRNA splicing complex in DDR. Our studies revealed that HDAC8 interacts with U2AF1 and modulates U2AF1 acetylation following DNA damage and acetylation on U2AF1-K23 is important for HDAC8 binding. Consistent with the enhanced HDAC8 activity induced by CM, we show that CM also diminished U2AF1 acetylation and impaired the assembly of the BRCA1-mRNA splicing complex after IR. To examine the consequences in alternative splicing events upon IR, we sorted CM KI (n=5) or control (n=3) Lin-Sca1+Kit+ (LSK) cells and exposed them to IR (2.0 Gy) followed by RNA-seq. We identified a total of 829 splicing events in WT-IR vs WT-NIR (NIR: no IR) and 643 splicing events in CM-IR vs CM-NIR. Specifically, we found that skipped exon (SE) and retained intron (RI) events are selectively reduced in CM vs WT (SE events: 496 vs 653; RI events: 2 vs 26). Gene set enrichment analysis revealed striking differential enrichment in 38 BRCA1-related pathways, including regulation of response to DNA damage stimulus (NES: -1.0816 vs 0.8707), regulation of DNA repair (NES: -1.1557 vs 0.7729), cell cycle checkpoint signaling (NES: -0.9719 vs 0.7895), and recombinational repair (NES: -0.9215 vs 0.7299). Altogether, these studies reveal mechanistic insights into DNA damage induced regulation of post-translational acetylation of U2AF1 splicing factor and provide a mechanistic link to the dysregulated alternative splicing and impaired DNA damage response in inv(16) AML.

RNA recognition by Npl3p reveals U2 snRNA-binding compatible with a chaperone role during splicing

The conserved SR-like protein Npl3 promotes splicing of diverse pre-mRNAs. However, the RNA sequence(s) recognized by the RNA Recognition Motifs (RRM1 & RRM2) of Npl3 during the splicing reaction remain elusive. Here, we developed a split-iCRAC approach in yeast to uncover the consensus sequence bound to each RRM. High-resolution NMR structures show that RRM2 recognizes a 5´-GNGG-3´ motif leading to an unusual mille-feuille topology. These structures also reveal how RRM1 preferentially interacts with a CC-dinucleotide upstream of this motif, and how the inter-RRM linker and the region C-terminal to RRM2 contribute to cooperative RNA-binding. Structure-guided functional studies show that Npl3 genetically interacts with U2 snRNP specific factors and we provide evidence that Npl3 melts U2 snRNA stem-loop I, a prerequisite for U2/U6 duplex formation within the catalytic center of the Bact spliceosomal complex. Thus, our findings suggest an unanticipated RNA chaperoning role for Npl3 during spliceosome active site formation.

Deletions of singular U1 snRNA gene significantly interfere with transcription and 3'-end mRNA formation.

Small nuclear RNAs (snRNAs) are structural and functional cores of the spliceosome. In metazoan genomes, each snRNA has multiple copies/variants, up to hundreds in mammals. However, the expressions and functions of each copy/variant in one organism have not been systematically studied. Focus on U1 snRNA genes, we investigated all five copies in Drosophila melanogaster using two series of constructed strains. Analyses of transgenic flies that each have a U1 promoter-driven gfp revealed that U1:21D is the major and ubiquitously expressed copy, and the other four copies have specificities in developmental stages and tissues. Mutant strains that each have a precisely deleted copy of U1-gene exhibited various extents of defects in fly morphology or mobility, especially deletion of U1:82Eb. Interestingly, splicing was changed at limited levels in the deletion strains, while large amounts of differentially-expressed genes and alternative polyadenylation events were identified, showing preferences in the down-regulation of genes with 1-2 introns and selection of proximal sites for 3'-end polyadenylation. In vitro assays suggested that Drosophila U1 variants pulled down fewer SmD2 proteins compared to the canonical U1. This study demonstrates that all five U1-genes in Drosophila have physiological functions in development and play regulatory roles in transcription and 3'-end formation.

Inter-species association mapping links splice site evolution to METTL16 and SNRNP27K.

Eukaryotic genes are interrupted by introns that are removed from transcribed RNAs by splicing. Patterns of splicing complexity differ between species, but it is unclear how these differences arise. We used inter-species association mapping with Saccharomycotina species to correlate splicing signal phenotypes with the presence or absence of splicing factors. Here, we show that variation in 5' splice site sequence preferences correlate with the presence of the U6 snRNA N6-methyladenosine methyltransferase METTL16 and the splicing factor SNRNP27K. The greatest variation in 5' splice site sequence occurred at the +4 position and involved a preference switch between adenosine and uridine. Loss of METTL16 and SNRNP27K orthologs, or a single SNRNP27K methionine residue, was associated with a preference for +4 U. These findings are consistent with splicing analyses of mutants defective in either METTL16 or SNRNP27K orthologs and models derived from spliceosome structures, demonstrating that inter-species association mapping is a powerful orthogonal approach to molecular studies. We identified variation between species in the occurrence of two major classes of 5' splice sites, defined by distinct interaction potentials with U5 and U6 snRNAs, that correlates with intron number. We conclude that variation in concerted processes of 5' splice site selection by U6 snRNA is associated with evolutionary changes in splicing signal phenotypes.

Development of Engineered-U1 snRNA Therapies: Current Status.

Splicing of pre-mRNA is a crucial regulatory stage in the pathway of gene expression. The majority of human genes that encode proteins undergo alternative pre-mRNA splicing and mutations that affect splicing are more prevalent than previously thought. Targeting aberrant RNA(s) may thus provide an opportunity to correct faulty splicing and potentially treat numerous genetic disorders. To that purpose, the use of engineered U1 snRNA (either modified U1 snRNAs or exon-specific U1s-ExSpeU1s) has been applied as a potentially therapeutic strategy to correct splicing mutations, particularly those affecting the 5' splice-site (5'ss). Here we review and summarize a vast panoply of studies that used either modified U1 snRNAs or ExSpeU1s to mediate gene therapeutic correction of splicing defects underlying a considerable number of genetic diseases. We also focus on the pre-clinical validation of these therapeutic approaches both in vitro and in vivo, and summarize the main obstacles that need to be overcome to allow for their successful translation to clinic practice in the future.

LinearCoFold and LinearCoPartition: linear-time algorithms for secondary structure prediction of interacting RNA molecules.

Many RNAs function through RNA-RNA interactions. Fast and reliable RNA structure prediction with consideration of RNA-RNA interaction is useful, however, existing tools are either too simplistic or too slow. To address this issue, we present LinearCoFold, which approximates the complete minimum free energy structure of two strands in linear time, and LinearCoPartition, which approximates the cofolding partition function and base pairing probabilities in linear time. LinearCoFold and LinearCoPartition are orders of magnitude faster than RNAcofold. For example, on a sequence pair with combined length of 26,190nt, LinearCoFold is 86.8× faster than RNAcofold MFE mode, and LinearCoPartition is 642.3× faster than RNAcofold partition function mode. Surprisingly, LinearCoFold and LinearCoPartition's predictions have higher PPV and sensitivity of intermolecular base pairs. Furthermore, we apply LinearCoFold to predict the RNA-RNA interaction between SARS-CoV-2 genomic RNA (gRNA) and human U4 small nuclear RNA (snRNA), which has been experimentally studied, and observe that LinearCoFold's prediction correlates better with the wet lab results than RNAcofold's.

MRNA splicing is modulated by intronic microRNAs

Splicing of transcripts is catalyzed by the spliceosome, a mega-complex consisting of hundreds of proteins and five snRNAs, which employs direct interactions. When U1 snRNA forms high-affinity binding, namely more than eight base pairs, with the 5'SS, the result is usually a suppressing effect on the splicing activity. This likely occurs due to the inefficient unwinding of U1/5'SS base-pairing or other regulatory obstructions. Here, we show invitro and in patient-derived cell lines that pre-microRNAs can modulate the splicing reaction by interacting with U1 snRNA. This leads to reduced binding affinity to the 5'SS, and hence promotes the inclusion of exons containing 5'SS, despite sequence-based high affinity to U1. Application of the mechanism resulted in correction of the splicing defect in the disease-causing VCAN gene from an individual with Wagner syndrome. This pre-miRNA/U1 interaction can regulate the expression of alternatively spliced exons, thus extending the scope of mechanisms regulating splicing.

Mechanism of U6 snRNA oligouridylation by human TUT1

U6 snRNA is a catalytic RNA responsible for pre-mRNA splicing reactions and undergoes various post-transcriptional modifications during its maturation process. The 3'-oligouridylation of U6 snRNA by the terminal uridylyltransferase, TUT1, provides the Lsm-binding site in U6 snRNA for U4/U6 di-snRNP formation and this ensures pre-mRNA splicing. Here, we present the crystal structure of human TUT1 (hTUT1) complexed with U6 snRNA, representing the post-uridylation of U6 snRNA by hTUT1. The N-terminal ZF-RRM and catalytic palm clamp the single-stranded AUA motif between the 5'-short stem and the 3'-telestem of U6 snRNA, and the ZF-RRM specifically recognizes the AUA motif. The ZF and the fingers hold the telestem, and the 3'-end of U6 snRNA is placed in the catalytic pocket of the palm for oligouridylation. The oligouridylation of U6 snRNA depends on the internal four-adenosine tract in the 5'-part of the telestem of U6 snRNA, and hTUT1 adds uridines until the internal adenosine tract can form base-pairs with the 3'-oligouridine tract. Together, the recognition of the specific structure and sequence of U6 snRNA by the multi-domain TUT1 protein and the intrinsic sequence and structure of U6 snRNA ensure the oligouridylation of U6 snRNA.

Predictive biomarkers for colorectal cancer: a state-of-the-art systematic review

Introduction Colorectal cancer (CRC) poses a substantial health burden, with early detection paramount for improved prognosis. This study aims to evaluate potential CRC biomarkers and detection techniques. Materials and Methods This systematic review, reported in adherence to PRISMA Statement 2020 guidelines, collates the latest research on potential biomarkers and detection/prognosis methods for CRC, spanning the last decade. Results Out of the 38 included studies, diverse biomarkers and detection methods emerged, with DNA methylation markers like SFRP2 and SDC2, microRNAs including miR-1290, miR-506, and miR-4316, and serum and plasma markers such as NTS levels and U2 snRNA fragments standing out. Methylated cfDNA and m5C methylation alteration in immune cells of the blood, along with circular RNA, showed promise as diagnostic markers. Meanwhile, techniques involving extracellular vesicles and lateral flow immunoassays exhibited potential for swift and effective CRC screening. Discussion Our state-of-the-art review identifies potential biomarkers, including SFRP2, SDC2, miR-1290, miR-506, miR-4316, and U2 snRNA fragments, with significant potential in enhancing CRC detection. However, comprehensive validation studies and a rigorous evaluation of clinical utility and cost-effectiveness remain necessary before integration into routine clinical practice. Conclusion The findings emphasize the need for continued research into biomarkers and detection methods to improve patient outcomes.

Pathogenicity analysis and splicing rescue of a classical splice site variant (c.1343+1G>T) of CNOT1 gene associated with neurodevelopmental disorders.

Mutations in the CNOT1 gene lead to an incurable rare neurological disorder mainly manifested as a clinical spectrum of intellectual disability, developmental delay, seizures, and behavioral problems. In this study, we investigated a classical splice site variant of CNOT1 (c.1343+1G>T) associated with neurodevelopmental disorders, which was a master regulator, orchestrating gene expression, RNA deadenylation, and protein ubiquitination. To link CNOT1 dysfunction with the neurodevelopmental phenotype observed in a patient, in vitro minigene assay was used to verify the effect of CNOT1 gene splice site variant c.1343+1G>T on mRNA splicing. We also explored the impact of transient transfection introducing modified U1 snRNA on correcting the splicing variant. Through minigene expression in mammalian cells, we demonstrated that the variant induced complete exon 12 skipping, which explained the patient's clinical condition and provided additional genetic diagnosis evidence for the clinical significance of the variant. Moreover, we confirmed that the aberrant splice pattern could be partially corrected by the modified U1 snRNA at the mRNA level, which provided strong evidence for the therapeutic potential of modified U1 snRNA in neutralizing the hazardous effect of incorrect splicing patterns.

Consequences of polyploidy and divergence as revealed by cytogenetic mapping of tandem repeats in African clawed frogs (Xenopus, Pipidae)

Repetitive elements have been identified in several amphibian genomes using whole genome sequencing, but few studies have used cytogenetic mapping to visualize these elements in this vertebrate group. Here, we used fluorescence in situ hybridization and genomic data to map the U1 and U2 small nuclear RNAs and histone H3 in six species of African clawed frog (genus Xenopus), including, from subgenus Silurana, the diploid Xenopus tropicalis and its close allotetraploid relative X. calcaratus and, from subgenus Xenopus, the allotetraploid species X. pygmaeus, X. allofraseri, X. laevis, and X. muelleri. Results allowed us to qualitatively evaluate the relative roles of polyploidization and divergence in the evolution of repetitive elements because our focal species include allotetraploid species derived from two independent polyploidization events — one that is relatively young that gave rise to X. calcaratus and another that is older that gave rise to the other (older) allotetraploids. Our results demonstrated conserved loci number and position of signals in the species from subgenus Silurana; allotetraploid X. calcaratus has twice as many signals as diploid X. tropicalis. However, the content of repeats varied among the other allotetraploid species. We detected almost same number of signals in X. muelleri as in X. calcaratus and same number of signals in X. pygmaeus, X. allofraseri, X. laevis as in the diploid X. tropicalis. Overall, these results are consistent with the proposal that allopolyploidization duplicated these tandem repeats and that variation in their copy number was accumulated over time through reduction and expansion in a subset of the older allopolyploids.

Pre-mRNA splicing order is predetermined and maintains splicing fidelity across multi-intronic transcripts.

Combinatorially, intron excision within a given nascent transcript could proceed down any of thousands of paths, each of which would expose different dynamic landscapes of cis-elements and contribute to alternative splicing. In this study, we found that post-transcriptional multi-intron splicing order in human cells is largely predetermined, with most genes spliced in one or a few predominant orders. Strikingly, these orders were conserved across cell types and stages of motor neuron differentiation. Introns flanking alternatively spliced exons were frequently excised last, after their neighboring introns. Perturbations to the spliceosomal U2 snRNA altered the preferred splicing order of many genes, and these alterations were associated with the retention of other introns in the same transcript. In one gene, early removal of specific introns was sufficient to induce delayed excision of three proximal introns, and this delay was caused by two distinct cis-regulatory mechanisms. Together, our results demonstrate that multi-intron splicing order in human cells is predetermined, is influenced by a component of the spliceosome and ensures splicing fidelity across long pre-mRNAs.

The fission yeast methyl phosphate capping enzyme Bmc1 guides 2'-O-methylation of the U6 snRNA.

Splicing requires the tight coordination of dynamic spliceosomal RNAs and proteins. U6 is the only spliceosomal RNA transcribed by RNA Polymerase III and undergoes an extensive maturation process. In humans and fission yeast, this includes addition of a 5' γ-monomethyl phosphate cap by members of the Bin3/MePCE family as well as snoRNA guided 2'-O-methylation. Previously, we have shown that the Bin3/MePCE homolog Bmc1 is recruited to the S. pombe telomerase holoenzyme by the LARP7 family protein Pof8, where it acts in a catalytic-independent manner to protect the telomerase RNA and facilitate holoenzyme assembly. Here, we show that Bmc1 and Pof8 are required for the formation of a distinct U6 snRNP that promotes 2'-O-methylation of U6, and identify a non-canonical snoRNA that guides this methylation. We also show that the 5' γ-monomethyl phosphate capping activity of Bmc1 is not required for its role in promoting snoRNA guided 2'-O-methylation, and that this role relies on different regions of Pof8 from those required for Pof8 function in telomerase. Our results are consistent with a novel role for Bmc1/MePCE family members in stimulating 2'-O-methylation and a more general role for Bmc1 and Pof8 in guiding noncoding RNP assembly beyond the telomerase RNP.

Sequence variations affect the 5' splice site selection of plant introns.

Introns are noncoding sequences spliced out of pre-mRNAs by the spliceosome to produce mature mRNAs. The 5' ends of introns mostly begin with GU and have a conserved sequence motif of AG/GUAAGU that could base-pair with the core sequence of U1 snRNA of the spliceosome. Intriguingly, ∼ 1% of introns in various eukaryotic species begin with GC. This occurrence could cause misannotation of genes; however, the underlying splicing mechanism is unclear. We analyzed the sequences around the intron 5' splice site (ss) in Arabidopsis (Arabidopsis thaliana) and found sequences at the GC intron ss are much more stringent than those of GT introns. Mutational analysis at various positions of the intron 5' ss revealed that although mutations impair base pairing, different mutations at the same site can have different effects, suggesting that steric hindrance also affects splicing. Moreover, mutations of 5' ss often activate a hidden ss nearby. Our data suggest that the 5' ss is selected via a competition between the major ss and the nearby minor ss. This work not only provides insights into the splicing mechanism of intron 5' ss but also improves the accuracy of gene annotation and the study of the evolution of intron 5' ss.

Cloning and Functional Verification of Endogenous U6 Promoters for the Establishment of Efficient CRISPR/Cas9-Based Genome Editing in Castor (Ricinus communis).

Castor (Ricinus communis) seeds are rich in a type of hydroxy fatty acid called ricinoleic acid, which is in high demand for the production of plant-based plastics, lubricants, and hydraulic oils. However, the high content of ricin, a toxic protein, in these seeds has restricted further expansion in the area of castor cultivation. Therefore, the development of ricin-free castor is needed. Genome editing technology, although successfully applied in several plant species, is still in the developing stages in castor and awaits the identification of an endogenous U6 promoter with robust function. Here, we searched for U6 small nuclear RNA (snRNA) genes in the castor genome. This led to the identification of six U6 snRNA genes. The promoters of these U6 snRNA genes were cloned, and their function was examined in castor cells using the particle delivery method. The results showed that a U6 promoter length of approximately 300 bp from the transcription start site was sufficient to activate gene expression. This study provides insights into the endogenous castor U6 promoter sequences and outlines a method for verifying the function of U6 promoters in plants using the particle delivery system.

A restrictor complex of ZC3H4, WDR82, and ARS2 integrates with PNUTS to control unproductive transcription

The transcriptional termination of unstable non-coding RNAs (ncRNAs) is poorly understood compared to coding transcripts. We recently identified ZC3H4-WDR82 ("restrictor") as restricting human ncRNA transcription, but how it does this is unknown. Here, we show that ZC3H4 additionally associates with ARS2 and the nuclear exosome targeting complex. The domains of ZC3H4 that contact ARS2 and WDR82 are required for ncRNA restriction, suggesting their presence in a functional complex. Consistently, ZC3H4, WDR82, and ARS2 co-transcriptionally control an overlapping population of ncRNAs. ZC3H4 is proximal to the negative elongation factor, PNUTS, which we show enables restrictor function and is required to terminate the transcription of all major RNA polymerase II transcript classes. In contrast to short ncRNAs, longer protein-coding transcription is supported by U1 snRNA, which shields transcripts from restrictor and PNUTS at hundreds of genes. These data provide important insights into the mechanism and control of transcription by restrictor and PNUTS.