An interpretable model of pre-mRNA splicing for animal and plant genes.

Pre-mRNA splicing is a fundamental step in gene expression, conserved across eukaryotes, in which the spliceosome recognizes motifs at the 3' and 5' splice sites (SSs), excises introns, and ligates exons. SS recognition and pairing is often influenced by protein splicing factors (SFs) that bind to splicing regulatory elements (SREs). Here, we describe SMsplice, a fully interpretable model of pre-mRNA splicing that combines models of core SS motifs, SREs, and exonic and intronic length preferences. We learn models that predict SS locations with 83 to 86% accuracy in fish, insects, and plants and about 70% in mammals. Learned SRE motifs include both known SF binding motifs and unfamiliar motifs, and both motif classes are supported by genetic analyses. Our comparisons across species highlight similarities between non-mammals, increased reliance on intronic SREs in plant splicing, and a greater reliance on SREs in mammalian splicing.

Biological relevance of alternative splicing in hematologic malignancies.

Alternative splicing (AS) is a strictly regulated process that generates multiple mRNA variants from a single gene, thus contributing to proteome diversity. Transcriptome-wide sequencing studies revealed networks of functionally coordinated splicing events, which produce isoforms with distinct or even opposing functions. To date, several mechanisms of AS are deregulated in leukemic cells, mainly due to mutations in splicing and/or epigenetic regulators and altered expression of splicing factors (SFs). In this review, we discuss aberrant splicing events induced by mutations affecting SFs (SF3B1, U2AF1, SRSR2, and ZRSR2), spliceosome components (PRPF8, LUC7L2, DDX41, and HNRNPH1), and epigenetic modulators (IDH1 and IDH2). Finally, we provide an extensive overview of the biological relevance of aberrant isoforms of genes involved in the regulation of apoptosis (e. g. BCL-X, MCL-1, FAS, and c-FLIP), activation of key cellular signaling pathways (CASP8, MAP3K7, and NOTCH2), and cell metabolism (PKM).

Truncating the spliceosomal 'rope protein' Prp45 results in Htz1 dependent phenotypes.

Spliceosome assembly contributes an important but incompletely understood aspect of splicing regulation. Prp45 is a yeast splicing factor which runs as an extended fold through the spliceosome, and which may be important for bringing its components together. We performed a whole genome analysis of the genetic interaction network of the truncated allele of PRP45 (prp45(1-169)) using synthetic genetic array technology and found chromatin remodellers and modifiers as an enriched category. In agreement with related studies, H2A.Z-encoding HTZ1, and the components of SWR1, INO80, and SAGA complexes represented prominent interactors, with htz1 conferring the strongest growth defect. Because the truncation of Prp45 disproportionately affected low copy number transcripts of intron-containing genes, we prepared strains carrying intronless versions of SRB2, VPS75, or HRB1, the most affected cases with transcription-related function. Intron removal from SRB2, but not from the other genes, partly repaired some but not all the growth phenotypes identified in the genetic screen. The interaction of prp45(1-169) and htz1Δ was detectable even in cells with SRB2 intron deleted (srb2Δi). The less truncated variant, prp45(1-330), had a synthetic growth defect with htz1Δ at 16°C, which also persisted in the srb2Δi background. Moreover, htz1Δ enhanced prp45(1-330) dependent pre-mRNA hyper-accumulation of both high and low efficiency splicers, genes ECM33 and COF1, respectively. We conclude that while the expression defects of low expression intron-containing genes contribute to the genetic interactome of prp45(1-169), the genetic interactions between prp45 and htz1 alleles demonstrate the sensitivity of spliceosome assembly, delayed in prp45(1-169), to the chromatin environment.

Intron lariat spliceosomes convert lariats to true circles: implications for intron transposition.

Rare, full-length circular intron RNAs distinct from lariats have been reported in several species, but their biogenesis is not understood. We envisioned and tested a hypothesis for their formation using Saccharomyces cerevisiae, documenting full-length and novel processed circular RNAs from multiple introns. Evidence implicates a previously undescribed catalytic activity of the intron lariat spliceosome (ILS) in which the 3'-OH of the lariat tail (with optional trimming and adenylation by the nuclear 3' processing machinery) attacks the branch, joining the intron 3' end to the 5' splice site in a 3'-5' linked circle. Human U2 and U12 spliceosomes produce analogous full-length and processed circles. Postsplicing catalytic activity of the spliceosome may promote intron transposition during eukaryotic genome evolution.

PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5´-splice-site selection causing tissue-specific defects.

The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5'-splice site (5'SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5'SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.

Dysfunction of Gpl1-Gih35-Wdr83 Complex in S. pombe Affects the Splicing of DNA Damage Repair Factors Resulting in Increased Sensitivity to DNA Damage.

Pre-mRNA splicing plays a key role in the regulation of gene expression. Recent discoveries suggest that defects in pre-mRNA splicing, resulting from the dysfunction of certain splicing factors, can impact the expression of genes crucial for genome surveillance mechanisms, including those involved in cellular response to DNA damage. In this study, we analyzed how cells with a non-functional spliceosome-associated Gpl1-Gih35-Wdr83 complex respond to DNA damage. Additionally, we investigated the role of this complex in regulating the splicing of factors involved in DNA damage repair. Our findings reveal that the deletion of any component within the Gpl1-Gih35-Wdr83 complex leads to a significant accumulation of unspliced pre-mRNAs of DNA repair factors. Consequently, mutant cells lacking this complex exhibit increased sensitivity to DNA-damaging agents. These results highlight the importance of the Gpl1-Gih35-Wdr83 complex in regulating the expression of DNA repair factors, thereby protecting the stability of the genome following DNA damage.

[Targeting the spliceosome: A new therapeutic strategy to counteract chemotherapy resistance in lung cancer?] / Cibler le splicéosome : une nouvelle stratégie thérapeutique pour contrecarrer la résistance à la chimiothérapie dans les cancers du poumon ?

Lung cancer is the first cancer-related cause of death worldwide. This is in partially due to therapeutic resistance, which occurs in around 70% of patients, especially those receiving platinum salts, the gold-standard chemotherapy. The massive deregulation of alternative transcript splicing processes observed in many cancers has led to the development of a new class of pharmacological agents aimed at inhibiting the activity of the splicing machinery (spliceosome). The molecular mechanisms by which these inhibitors act remain largely unknown, as do the benefits of using them in combination with other therapies. In this context, our work is focused on an inhibitor of the SRPK1 kinase, a major regulator of the spliceosome.

A role for SNU66 in maintaining 5' splice site identity during spliceosome assembly.

In spliceosome assembly, the 5' splice site is initially recognized by U1 snRNA. U1 leaves the spliceosome during the assembly process, therefore other factors contribute to the maintenance of 5' splice site identity as it is loaded into the catalytic site. Recent structural data suggest that human tri-snRNP 27K (SNRP27) M141 and SNU66 H734 interact to stabilize the U4/U6 quasi-pseudo knot at the base of the U6 snRNA ACAGAGA box in pre-B complex. Previously, we found that mutations in Caenorhabditis elegans at SNRP-27 M141 promote changes in alternative 5'ss usage. We tested whether the potential interaction between SNRP-27 M141 and SNU-66 H765 (the C. elegans equivalent position to human SNU66 H734) contributes to maintaining 5' splice site identity during spliceosome assembly. We find that SNU-66 H765 mutants promote alternative 5' splice site usage. Many of the alternative 5' splicing events affected by SNU-66(H765G) overlap with those affected SNRP-27(M141T). Double mutants of snrp-27(M141T) and snu-66(H765G) are homozygous lethal. We hypothesize that mutations at either SNRP-27 M141 or SNU-66 H765 allow the spliceosome to load alternative 5' splice sites into the active site. Tests with mutant U1 snRNA and swapped 5' splice sites indicate that the ability of SNRP-27 M141 and SNU-66 H765 mutants to affect a particular 5' splice alternative splicing event is dependent on both the presence of a weaker consensus 5'ss nearby and potentially nearby splicing factor binding sites. Our findings confirm a new role for the C terminus of SNU-66 in maintenance of 5' splice site identity during spliceosome assembly.

Unconventional features in the transcription and processing of spliceosomal small nuclear RNAs in the protozoan parasite Trichomonas vaginalis.

Trichomonas vaginalis is a medically important protozoan parasite, and a deep-branching, evolutionarily divergent unicellular eukaryote that has conserved several key features of eukaryotic gene expression. Trichomonas vaginalis possesses a metazoan/plant-like capping apparatus, mRNAs with a cap 1 structure and spliceosomes containing the five small nuclear RNAs (snRNAs). However, in contrast to metazoan and plant snRNAs, the structurally conserved T. vaginalis snRNAs were initially identified as lacking the canonical guanosine cap nucleotide. To explain this unusual condition, we sought to investigate transcriptional and processing features of the spliceosomal snRNAs in this protist. Here, we show that T. vaginalis spliceosomal snRNA genes mostly lack typical eukaryotic promoters. In contrast to other eukaryotes, the putative TATA box in the T. vaginalis U6 snRNA gene was found to be dispensable for transcription or RNA polymerase selectivity. Moreover, U6 transcription in T. vaginalis was virtually insensitive to tagetitoxin compared with other cellular transcripts produced by the same RNA polymerase III. Most important and unexpected, snRNA transcription in T. vaginalis appears to bypass capping as we show that these transcripts retain their original 5'-triphosphate groups. In conclusion, transcription and processing of spliceosomal snRNAs in T. vaginalis deviate considerably from the conventional rules of other eukaryotes.

Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome.

Here, we identify RBM41 as a novel unique protein component of the minor spliceosome. RBM41 has no previously recognized cellular function but has been identified as a paralog of U11/U12-65K, a known unique component of the U11/U12 di-snRNP. Both proteins use their highly similar C-terminal RRMs to bind to 3'-terminal stem-loops in U12 and U6atac snRNAs with comparable affinity. Our BioID data indicate that the unique N-terminal domain of RBM41 is necessary for its association with complexes containing DHX8, an RNA helicase, which in the major spliceosome drives the release of mature mRNA from the spliceosome. Consistently, we show that RBM41 associates with excised U12-type intron lariats, is present in the U12 mono-snRNP, and is enriched in Cajal bodies, together suggesting that RBM41 functions in the post-splicing steps of the minor spliceosome assembly/disassembly cycle. This contrasts with U11/U12-65K, which uses its N-terminal region to interact with U11 snRNP during intron recognition. Finally, while RBM41 knockout cells are viable, they show alterations in U12-type 3' splice site usage. Together, our results highlight the role of the 3'-terminal stem-loop of U12 snRNA as a dynamic binding platform for the U11/U12-65K and RBM41 proteins, which function at distinct stages of the assembly/disassembly cycle.

Tracing Allostery in the Spliceosome Ski2-like RNA Helicase Brr2.

RNA ATPases/helicases remodel substrate RNA-protein complexes in distinct ways. The different RNA ATPases/helicases, taking part in the spliceosome complex, reshape the RNA/RNA-protein contacts to enable premature-mRNA splicing. Among them, the bad response to refrigeration 2 (Brr2) helicase promotes U4/U6 small nuclear (sn)RNA unwinding via ATP-driven translocation of the U4 snRNA strand, thus playing a pivotal role during the activation, catalytic, and disassembly phases of splicing. The plastic Brr2 architecture consists of an enzymatically active N-terminal cassette (N-cassette) and a structurally similar but inactive C-terminal cassette (C-cassette). The C-cassette, along with other allosteric effectors and regulators, tightly and timely controls Brr2's function via an elusive mechanism. Here, microsecond-long molecular dynamics simulations, dynamical network theory, and community network analysis are combined to elucidate how allosteric effectors/regulators modulate the Brr2 function. We unexpectedly reveal that U4 snRNA itself acts as an allosteric regulator, amplifying the cross-talk of distal Brr2 domains and triggering a conformational reorganization of the protein. Our findings offer fundamental understanding into Brr2's mechanism of action and broaden our knowledge on the sophisticated regulatory mechanisms by which spliceosome ATPases/helicases control gene expression. This includes their allosteric regulation exerted by client RNA strands, a mechanism that may be broadly applicable to other RNA-dependent ATPases/helicases.

The splicing regulators RBM5 and RBM10 are subunits of the U2 snRNP engaged with intron branch sites on chromatin.

Understanding the mechanisms of pre-mRNA splicing is limited by the technical challenges to examining spliceosomes in vivo. Here, we report the isolation of RNP complexes derived from precatalytic A or B-like spliceosomes solubilized from the chromatin pellet of mammalian cell nuclei. We found that these complexes contain U2 snRNP proteins and a portion of the U2 snRNA bound with protected RNA fragments that precisely map to intronic branch sites across the transcriptome. These U2 complexes also contained the splicing regulators RBM5 and RBM10. We found RBM5 and RBM10 bound to nearly all branch site complexes and not simply those at regulated exons. The deletion of a conserved RBM5/RBM10 peptide sequence, including a zinc finger motif, disrupted U2 interaction and rendered the proteins inactive for the repression of many alternative exons. We propose a model where RBM5 and RBM10 regulate splicing as components of the U2 snRNP complex following branch site base pairing.

The spliceosome-associated protein CWC15 promotes miRNA biogenesis in Arabidopsis.

MicroRNAs (miRNAs) play a key role in regulating gene expression and their biogenesis is precisely controlled through modulating the activity of microprocessor. Here, we report that CWC15, a spliceosome-associated protein, acts as a positive regulator of miRNA biogenesis. CWC15 binds the promoters of genes encoding miRNAs (MIRs), promotes their activity, and increases the occupancy of DNA-dependent RNA polymerases at MIR promoters, suggesting that CWC15 positively regulates the transcription of primary miRNA transcripts (pri-miRNAs). In addition, CWC15 interacts with Serrate (SE) and HYL1, two key components of microprocessor, and is required for efficient pri-miRNA processing and the HYL1-pri-miRNA interaction. Moreover, CWC15 interacts with the 20 S proteasome and PRP4KA, facilitating SE phosphorylation by PRP4KA, and subsequent non-functional SE degradation by the 20 S proteasome. These data reveal that CWC15 ensures optimal miRNA biogenesis by maintaining proper SE levels and by modulating pri-miRNA levels. Taken together, this study uncovers the role of a conserved splicing-related protein in miRNA biogenesis.

Minor introns impact on hematopoietic malignancies.

In the intricate orchestration of the central dogma, pre-mRNA splicing plays a crucial role in the post-transcriptional process that transforms DNA into mature mRNA. Widely acknowledged as a pivotal RNA processing step, it significantly influences gene expression and alters the functionality of gene product proteins. Although U2-dependent spliceosomes efficiently manage the removal of over 99% of introns, a distinct subset of essential genes undergo splicing with a different intron type, denoted as minor introns, using U12-dependent spliceosomes. Mutations in spliceosome component genes are now recognized as prevalent genetic abnormalities in cancer patients, especially those with hematologic malignancies. Despite the relative rarity of minor introns, genes containing them are evolutionarily conserved and play crucial roles in functions such as the RAS-MAPK pathway. Disruptions in U12-type minor intron splicing caused by mutations in snRNA or its regulatory components significantly contribute to cancer progression. Notably, recurrent mutations associated with myelodysplastic syndrome (MDS) in the minor spliceosome component ZRSR2 underscore its significance. Examination of ZRSR2-mutated MDS cells has revealed that only a subset of minor spliceosome-dependent genes, such as LZTR1, consistently exhibit missplicing. Recent technological advancements have uncovered insights into minor introns, raising inquiries beyond current understanding. This review comprehensively explores the importance of minor intron regulation, the molecular implications of minor (U12-type) spliceosomal mutations and cis-regulatory regions, and the evolutionary progress of studies on minor, aiming to provide a sophisticated understanding of their intricate role in cancer biology.

Cryo-EM analyses of dimerized spliceosomes provide new insights into the functions of B complex proteins.

The B complex is a key intermediate stage of spliceosome assembly. To improve the structural resolution of monomeric, human spliceosomal B (hB) complexes and thereby generate a more comprehensive hB molecular model, we determined the cryo-EM structure of B complex dimers formed in the presence of ATP γ S. The enhanced resolution of these complexes allows a finer molecular dissection of how the 5' splice site (5'ss) is recognized in hB, and new insights into molecular interactions of FBP21, SNU23 and PRP38 with the U6/5'ss helix and with each other. It also reveals that SMU1 and RED are present as a heterotetrameric complex and are located at the interface of the B dimer protomers. We further show that MFAP1 and UBL5 form a 5' exon binding channel in hB, and elucidate the molecular contacts stabilizing the 5' exon at this stage. Our studies thus yield more accurate models of protein and RNA components of hB complexes. They further allow the localization of additional proteins and protein domains (such as SF3B6, BUD31 and TCERG1) whose position was not previously known, thereby uncovering new functions for B-specific and other hB proteins during pre-mRNA splicing.

SNUPN deficiency causes a recessive muscular dystrophy due to RNA mis-splicing and ECM dysregulation.

SNURPORTIN-1, encoded by SNUPN, plays a central role in the nuclear import of spliceosomal small nuclear ribonucleoproteins. However, its physiological function remains unexplored. In this study, we investigate 18 children from 15 unrelated families who present with atypical muscular dystrophy and neurological defects. Nine hypomorphic SNUPN biallelic variants, predominantly clustered in the last coding exon, are ascertained to segregate with the disease. We demonstrate that mutant SPN1 failed to oligomerize leading to cytoplasmic aggregation in patients' primary fibroblasts and CRISPR/Cas9-mediated mutant cell lines. Additionally, mutant nuclei exhibit defective spliceosomal maturation and breakdown of Cajal bodies. Transcriptome analyses reveal splicing and mRNA expression dysregulation, particularly in sarcolemmal components, causing disruption of cytoskeletal organization in mutant cells and patient muscle tissues. Our findings establish SNUPN deficiency as the genetic etiology of a previously unrecognized subtype of muscular dystrophy and provide robust evidence of the role of SPN1 for muscle homeostasis.

Xrp1 governs the stress response program to spliceosome dysfunction.

Co-transcriptional processing of nascent pre-mRNAs by the spliceosome is vital to regulating gene expression and maintaining genome integrity. Here, we show that the deficiency of functional U5 small nuclear ribonucleoprotein particles (snRNPs) in Drosophila imaginal cells causes extensive transcriptome remodeling and accumulation of highly mutagenic R-loops, triggering a robust stress response and cell cycle arrest. Despite compromised proliferative capacity, the U5 snRNP-deficient cells increased protein translation and cell size, causing intra-organ growth disbalance before being gradually eliminated via apoptosis. We identify the Xrp1-Irbp18 heterodimer as the primary driver of transcriptional and cellular stress program downstream of U5 snRNP malfunction. Knockdown of Xrp1 or Irbp18 in U5 snRNP-deficient cells attenuated JNK and p53 activity, restored normal cell cycle progression and growth, and inhibited cell death. Reducing Xrp1-Irbp18, however, did not rescue the splicing defects, highlighting the requirement of accurate splicing for cellular and tissue homeostasis. Our work provides novel insights into the crosstalk between splicing and the DNA damage response and defines the Xrp1-Irbp18 heterodimer as a critical sensor of spliceosome malfunction and mediator of the stress-induced cellular senescence program.

The removal of introns and the joining of exons into mature mRNA by the spliceosome is crucial in regulating gene expression, simultaneously safeguarding genome integrity and enhancing proteome diversity in multicellular organisms. Spliceosome dysfunction is thus associated with various diseases and organismal aging. Our study describes the cascade of events in response to spliceosome dysfunction. We identified two transcription factors as drivers of a stress response program triggered by spliceosome dysfunction, which dramatically remodel gene expression to protect tissue integrity and induce a senescent-like state in damaged cells prior to their inevitable elimination. Together, we highlight the indispensable role of spliceosomes in maintaining homeostasis and implicate spliceosome dysfunction in senescent cell accumulation associated with the pathomechanisms of spliceopathies and aging.

Spliceosomal helicases DDX41/SACY-1 and PRP22/MOG-5 both contribute to proofreading against proximal 3' splice site usage.

RNA helicases drive necessary rearrangements and ensure fidelity during the pre-mRNA splicing cycle. DEAD-box helicase DDX41 has been linked to human disease and has recently been shown to interact with DEAH-box helicase PRP22 in the spliceosomal C* complex, yet its function in splicing remains unknown. Depletion of DDX41 homolog SACY-1 from somatic cells has been previously shown to lead to changes in alternative 3' splice site (3'ss) usage. Here, we show by transcriptomic analysis of published and novel data sets that SACY-1 perturbation causes a previously unreported pattern in alternative 3' splicing in introns with pairs of 3' splice sites ≤18 nt away from each other. We find that both SACY-1 depletion and the allele sacy-1(G533R) lead to a striking unidirectional increase in the usage of the proximal (upstream) 3'ss. We previously discovered a similar alternative splicing pattern between germline tissue and somatic tissue, in which there is a unidirectional increase in proximal 3'ss usage in the germline for ∼200 events; many of the somatic SACY-1 alternative 3' splicing events overlap with these developmentally regulated events. We generated targeted mutant alleles of the Caenorhabditis elegans homolog of PRP22, mog-5, in the region of MOG-5 that is predicted to interact with SACY-1 based on the human C* structure. These viable alleles, and a mimic of the myelodysplastic syndrome-associated allele DDX41(R525H), all promote the usage of proximal alternative adjacent 3' splice sites. We show that PRP22/MOG-5 and DDX41/SACY-1 have overlapping roles in proofreading the 3'ss.

THUMPD2 catalyzes the N2-methylation of U6 snRNA of the spliceosome catalytic center and regulates pre-mRNA splicing and retinal degeneration.

The mechanisms by which the relatively conserved spliceosome manages the enormously large number of splicing events that occur in humans (∼200 000 versus ∼300 in yeast) are poorly understood. Here, we show deposition of one RNA modification-N2-methylguanosine (m2G) on the G72 of U6 snRNA (the catalytic center of the spliceosome) promotes efficient pre-mRNA splicing activity in human cells. This modification was identified to be conserved among vertebrates. Further, THUMPD2 was demonstrated as the methyltransferase responsible for U6 m2G72 by explicitly recognizing the U6-specific sequences and structural elements. The knock-out of THUMPD2 eliminated U6 m2G72 and impaired the pre-mRNA splicing activity, resulting in thousands of changed alternative splicing events of endogenous pre-mRNAs in human cells. Notably, the aberrantly spliced pre-mRNA population elicited the nonsense-mediated mRNA decay pathway. We further show that THUMPD2 was associated with age-related macular degeneration and retinal function. Our study thus demonstrates how an RNA epigenetic modification of the major spliceosome regulates global pre-mRNA splicing and impacts physiology and disease.

RIP-PEN-seq identifies a class of kink-turn RNAs as splicing regulators.

A kink-turn (K-turn) is a three-dimensional RNA structure that exists in all three primary phylogenetic domains. In this study, we developed the RIP-PEN-seq method to identify the full-length sequences of RNAs bound by the K-turn binding protein 15.5K and discovered a previously uncharacterized class of RNAs with backward K-turn motifs (bktRNAs) in humans and mice. All bktRNAs share two consensus sequence motifs at their fixed terminal position and have complex folding properties, expression and evolution patterns. We found that a highly conserved bktRNA1 guides the methyltransferase fibrillarin to install RNA methylation of U12 small nuclear RNA in humans. Depletion of bktRNA1 causes global splicing dysregulation of U12-type introns by impairing the recruitment of ZCRB1 to the minor spliceosome. Most bktRNAs regulate the splicing of local introns by interacting with the 15.5K protein. Taken together, our findings characterize a class of small RNAs and uncover another layer of gene expression regulation that involves crosstalk among bktRNAs, RNA splicing and RNA methylation.