End Cleavage Research Articles

Alternative cleavage and polyadenylation in health and disease.

Most human genes have multiple sites at which RNA 3' end cleavage and polyadenylation can occur, enabling the expression of distinct transcript isoforms under different conditions. Novel methods to sequence RNA 3' ends have generated comprehensive catalogues of polyadenylation (poly(A)) sites; their analysis using innovative computational methods has revealed how poly(A) site choice is regulated by core RNA 3' end processing factors, such as cleavage factor I and cleavage and polyadenylation specificity factor, as well as by other RNA-binding proteins, particularly splicing factors. Here, we review the experimental and computational methods that have enabled the global mapping of mRNA and of long non-coding RNA 3' ends, quantification of the resulting isoforms and the discovery of regulators of alternative cleavage and polyadenylation (APA). We highlight the different types of APA-derived isoforms and their functional differences, and illustrate how APA contributes to human diseases, including cancer and haematological, immunological and neurological diseases.

Nuclear eIF4E Stimulates 3′-End Cleavage of Target RNAs

SUMMARYThe eukaryotic translation initiation factor eIF4E is nuclear and cytoplasmic where it plays roles in export and translation of specific transcripts, respectively. When we were studying its mRNA export activity, we unexpectedly discovered that eIF4E drives the protein expression of elements of the 3′-end core cleavage complex involved in cleavage and polyadenylation (CPA), including CPSF3, the enzyme responsible for cleavage, as well as its co-factors CPSF1, CPSF2, CPSF4, Symplekin, WDR33, and FIP1L1. Using multiple strategies, we demonstrate that eIF4E stimulates 3′-end cleavage of selected RNAs. eIF4E physically interacts with CPSF3, CPSF1, and uncleaved target RNA, suggesting it acts directly and indirectly on the pathway. Through these effects, eIF4E can generate better substrates for its mRNA export and translation activities. Thus, we identified an unanticipated function for eIF4E in 3′-end processing of specific target RNAs, and this function could potentially affect the expression of a broad range of oncoproteins.

U1 snRNP Telescripting: Suppression of Premature Transcription Termination in Introns as a New Layer of Gene Regulation.

Recent observations showed that nascent RNA polymerase II transcripts, pre-mRNAs, and noncoding RNAs are highly susceptible to premature 3'-end cleavage and polyadenylation (PCPA) from numerous intronic cryptic polyadenylation signals (PASs). The importance of this in gene regulation was not previously appreciated as PASs, despite their prevalence, were thought to be active in terminal exons at gene ends. Unexpectedly, antisense oligonucleotide interference with U1 snRNA base-pairing to 5' splice sites, which is necessary for U1 snRNP's (U1) function in splicing, caused widespread PCPA in metazoans. This uncovered U1's PCPA suppression activity, termed telescripting, as crucial for full-length transcription in thousands of vertebrate genes, providing a general role in transcription elongation control. Progressive intron-size expansion in metazoan evolution greatly increased PCPA vulnerability and dependence on U1 telescripting. We describe how these observations unfolded and discuss U1 telescripting's role in shaping the transcriptome.

U1 snRNP Telescripting Roles in Transcription and Its Mechanism.

Telescripting is a fundamental cotranscriptional gene regulation process that relies on U1 snRNP (U1) to suppress premature 3'-end cleavage and polyadenylation (PCPA) in RNA polymerase II (Pol II) transcripts, which is necessary for full-length transcription of thousands of protein-coding (pre-mRNAs) and long noncoding (lncRNA) genes. Like U1 role in splicing, telescripting requires U1 snRNA base-pairing with nascent transcripts. Inhibition of U1 base-pairing with U1 snRNA antisense morpholino oligonucleotide (U1 AMO) mimics widespread PCPA from cryptic polyadenylation signals (PASs) in human tissues, including PCPA in introns and last exons' 3'-untranslated regions (3' UTRs). U1 telescripting-PCPA balance changes generate diverse RNAs depending on where in a gene it occurs. Long genes are highly U1-telescripting-dependent because of PASs in introns compared to short genes. Enrichment of cell cycle control, differentiation, and developmental functions in long genes, compared to housekeeping and acute cell stress response genes in short genes, reveals a gene size-function relationship in mammalian genomes. This polarization increased in metazoan evolution by previously unexplained intron expansion, suggesting that U1 telescripting could shift global gene expression priorities. We show that that modulating U1 availability can profoundly alter cell phenotype, such as cancer cell migration and invasion, underscoring the critical role of U1 homeostasis and suggesting it as a potential target for therapies. We describe a complex of U1 with cleavage and polyadenylation factors that silences PASs in introns and 3' UTR, which gives insights into U1 telescripting mechanism and transcription elongation regulation.

Structural insight into the human mitochondrial tRNA purine N1-methyltransferase and ribonuclease P complexes

Mitochondrial tRNAs are transcribed as long polycistronic transcripts of precursor tRNAs and undergo posttranscriptional modifications such as endonucleolytic processing and methylation required for their correct structure and function. Among them, 5′-end processing and purine 9 N1-methylation of mitochondrial tRNA are catalyzed by two proteinaceous complexes with overlapping subunit composition. The Mg2+-dependent RNase P complex for 5′-end cleavage comprises the methyltransferase domain–containing protein tRNA methyltransferase 10C, mitochondrial RNase P subunit (TRMT10C/MRPP1), short-chain oxidoreductase hydroxysteroid 17β-dehydrogenase 10 (HSD17B10/MRPP2), and metallonuclease KIAA0391/MRPP3. An MRPP1–MRPP2 subcomplex also catalyzes the formation of 1-methyladenosine/1-methylguanosine at position 9 using S-adenosyl-l-methionine as methyl donor. However, a lack of structural information has precluded insights into how these complexes methylate and process mitochondrial tRNA. Here, we used a combination of X-ray crystallography, interaction and activity assays, and small angle X-ray scattering (SAXS) to gain structural insight into the two tRNA modification complexes and their components. The MRPP1 N terminus is involved in tRNA binding and monomer–monomer self-interaction, whereas the C-terminal SPOUT fold contains key residues for S-adenosyl-l-methionine binding and N1-methylation. The entirety of MRPP1 interacts with MRPP2 to form the N1-methylation complex, whereas the MRPP1–MRPP2–MRPP3 RNase P complex only assembles in the presence of precursor tRNA. This study proposes low-resolution models of the MRPP1–MRPP2 and MRPP1–MRPP2–MRPP3 complexes that suggest the overall architecture, stoichiometry, and orientation of subunits and tRNA substrates.

SWI/SNF interacts with cleavage and polyadenylation factors and facilitates pre-mRNA 3' end processing.

SWI/SNF complexes associate with genes and regulate transcription by altering the chromatin at the promoter. It has recently been shown that these complexes play a role in pre-mRNA processing by associating at alternative splice sites. Here, we show that SWI/SNF complexes are involved also in pre-mRNA 3′ end maturation by facilitating 3′ end cleavage of specific pre-mRNAs. Comparative proteomics show that SWI/SNF ATPases interact physically with subunits of the cleavage and polyadenylation complexes in fly and human cells. In Drosophila melanogaster, the SWI/SNF ATPase Brahma (dBRM) interacts with the CPSF6 subunit of cleavage factor I. We have investigated the function of dBRM in 3′ end formation in S2 cells by RNA interference, single-gene analysis and RNA sequencing. Our data show that dBRM facilitates pre-mRNA cleavage in two different ways: by promoting the association of CPSF6 to the cleavage region and by stabilizing positioned nucleosomes downstream of the cleavage site. These findings show that SWI/SNF complexes play a role also in the cleavage of specific pre-mRNAs in animal cells.

Protein composition of catalytically active U7-dependent processing complexes assembled on histone pre-mRNA containing biotin and a photo-cleavable linker.

3′ end cleavage of metazoan replication-dependent histone pre-mRNAs requires the multi-subunit holo-U7 snRNP and the stem–loop binding protein (SLBP). The exact composition of the U7 snRNP and details of SLBP function in processing remain unclear. To identify components of the U7 snRNP in an unbiased manner, we developed a novel approach for purifying processing complexes from Drosophila and mouse nuclear extracts. In this method, catalytically active processing complexes are assembled in vitro on a cleavage-resistant histone pre-mRNA containing biotin and a photo-sensitive linker, and eluted from streptavidin beads by UV irradiation for direct analysis by mass spectrometry. In the purified processing complexes, Drosophila and mouse U7 snRNP have a remarkably similar composition, always being associated with CPSF73, CPSF100, symplekin and CstF64. Many other proteins previously implicated in the U7-dependent processing are not present. Drosophila U7 snRNP bound to histone pre-mRNA in the absence of SLBP contains the same subset of polyadenylation factors but is catalytically inactive and addition of recombinant SLBP is sufficient to trigger cleavage. This result suggests that Drosophila SLBP promotes a structural rearrangement of the processing complex, resulting in juxtaposition of the CPSF73 endonuclease with the cleavage site in the pre-mRNA substrate.

Xrn2 accelerates termination by RNA polymerase II, which is underpinned by CPSF73 activity.

Termination is a ubiquitous phase in every transcription cycle but is incompletely understood and a subject of debate. We used gene editing as a new approach to address its mechanism through engineered conditional depletion of the 5' → 3' exonuclease Xrn2 or the polyadenylation signal (PAS) endonuclease CPSF73 (cleavage and polyadenylation specificity factor 73). The ability to rapidly control Xrn2 reveals a clear and general role for it in cotranscriptional degradation of 3' flanking region RNA and transcriptional termination. This defect is characterized genome-wide at high resolution using mammalian native elongating transcript sequencing (mNET-seq). An Xrn2 effect on termination requires prior RNA cleavage, and we provide evidence for this by showing that catalytically inactive CPSF73 cannot restore termination to cells lacking functional CPSF73. Notably, Xrn2 plays no significant role in either Histone or small nuclear RNA (snRNA) gene termination even though both RNA classes undergo 3' end cleavage. In sum, efficient termination on most protein-coding genes involves CPSF73-mediated RNA cleavage and cotranscriptional degradation of polymerase-associated RNA by Xrn2. However, as CPSF73 loss caused more extensive readthrough transcription than Xrn2 elimination, it likely plays a more underpinning role in termination.

Structural basis for DNA 3\u2032-end processing by human tyrosyl-DNA phosphodiesterase 1

Tyrosyl-DNA phosphodiesterase (Tdp1) is a DNA 3′-end processing enzyme that repairs topoisomerase 1B-induced DNA damage. We use a new tool combining site-specific DNA–protein cross-linking with mass spectrometry to identify Tdp1 interactions with DNA. A conserved phenylalanine (F259) of Tdp1, required for efficient DNA processing in biochemical assays, cross-links to defined positions in DNA substrates. Crystal structures of Tdp1–DNA complexes capture the DNA repair machinery after 3′-end cleavage; these reveal how Tdp1 coordinates the 3′-phosphorylated product of nucleosidase activity and accommodates duplex DNA. A hydrophobic wedge splits the DNA ends, directing the scissile strand through a channel towards the active site. The F259 side-chain stacks against the −3 base pair, delimiting the junction of duplexed and melted DNA, and fixes the scissile strand in the channel. Our results explain why Tdp1 cleavage is non-processive and provide a molecular basis for DNA 3′-end processing by Tdp1.

Cryo-EM structure of a late pre-40S ribosomal subunit from Saccharomyces cerevisiae.

Mechanistic understanding of eukaryotic ribosome formation requires a detailed structural knowledge of the numerous assembly intermediates, generated along a complex pathway. Here, we present the structure of a late pre-40S particle at 3.6 Å resolution, revealing in molecular detail how assembly factors regulate the timely folding of pre-18S rRNA. The structure shows that, rather than sterically blocking 40S translational active sites, the associated assembly factors Tsr1, Enp1, Rio2 and Pno1 collectively preclude their final maturation, thereby preventing untimely tRNA and mRNA binding and error prone translation. Moreover, the structure explains how Pno1 coordinates the 3'end cleavage of the 18S rRNA by Nob1 and how the late factor's removal in the cytoplasm ensures the structural integrity of the maturing 40S subunit.

Two distinct transcription termination modes dictated by promoters.

Transcription termination determines the ends of transcriptional units and thereby ensures the integrity of the transcriptome and faithful gene regulation. Studies in yeast and human cells have identified the exoribonuclease XRN2 as a key termination factor for protein-coding genes. Here we performed a genome-wide investigation of RNA polymerase II (Pol II) transcription termination in XRN2-deficient Caenorhabditis elegans and observed two distinct modes of termination. Although a subset of genes requires XRN2, termination of other genes appears both independent of, and refractory to, XRN2. XRN2 independence is not merely a consequence of failure to recruit XRN2, since XRN2 is present on-and promotes Pol II accumulation near the polyadenylation sites of-both gene classes. Unexpectedly, promoters instruct the choice of termination mode, but XRN2-independent termination additionally requires a compatible region downstream from the 3' end cleavage site. Hence, different termination mechanisms may work with different configurations of Pol II complexes dictated by promoters.

SnRNA 3' End Processing by a CPSF73-Containing Complex Essential for Development in Arabidopsis.

Uridine-rich small nuclear RNAs (snRNAs) are the basal components of the spliceosome and play essential roles in splicing. The biogenesis of the majority of snRNAs involves 3′ end endonucleolytic cleavage of the nascent transcript from the elongating DNA-dependent RNA ploymerase II. However, the protein factors responsible for this process remain elusive in plants. Here, we show that DEFECTIVE in snRNA PROCESSING 1 (DSP1) is an essential protein for snRNA 3′ end maturation in Arabidopsis. A hypomorphic dsp1-1 mutation causes pleiotropic developmental defects, impairs the 3′ end processing of snRNAs, increases the levels of snRNA primary transcripts (pre-snRNAs), and alters the occupancy of Pol II at snRNA loci. In addition, DSP1 binds snRNA loci and interacts with Pol-II in a DNA/RNA-dependent manner. We further show that DSP1 forms a conserved complex, which contains at least four additional proteins, to catalyze snRNA 3′ end maturation in Arabidopsis. The catalytic component of this complex is likely the cleavage and polyadenylation specificity factor 73 kDa-I (CSPF73-I), which is the nuclease cleaving the pre-mRNA 3′ end. However, the DSP1 complex does not affect pre-mRNA 3′ end cleavage, suggesting that plants may use different CPSF73-I-containing complexes to process snRNAs and pre-mRNAs. This study identifies a complex responsible for the snRNA 3′ end maturation in plants and uncovers a previously unknown function of CPSF73 in snRNA maturation.

A Hypertension-Associated tRNAAla Mutation Alters tRNA Metabolism and Mitochondrial Function.

In this report, we investigated the pathophysiology of a novel hypertension-associated mitochondrial tRNAAla 5655A → G (m.5655A → G) mutation. The destabilization of a highly conserved base pairing (A1-U72) at the aminoacyl acceptor stem by an m.5655A → G mutation altered the tRNAAla function. An in vitro processing analysis showed that the m.5655A → G mutation reduced the efficiency of tRNAAla precursor 5′ end cleavage catalyzed by RNase P. By using cybrids constructed by transferring mitochondria from lymphoblastoid cell lines derived from a Chinese family into mitochondrial DNA (mtDNA)-less (ρo) cells, we showed a 41% reduction in the steady-state level of tRNAAla in mutant cybrids. The mutation caused an improperly aminoacylated tRNAAla, as suggested by aberrantly aminoacylated tRNAAla and slower electrophoretic mobility of mutated tRNA. A failure in tRNAAla metabolism contributed to variable reductions in six mtDNA-encoded polypeptides in mutant cells, ranging from 21% to 37.5%, with an average of a 29.1% reduction, compared to levels of the controls. The impaired translation caused reduced activities of mitochondrial respiration chains. Furthermore, marked decreases in the levels of mitochondrial ATP and membrane potential were observed in mutant cells. These caused increases in the production of reactive oxygen species in the mutant cybrids. The data provide evidence for the association of the tRNAAla 5655A → G mutation with hypertension.

Role of miRNAs and alternative mRNA 3′-end cleavage and polyadenylation of their mRNA targets in cardiomyocyte hypertrophy

miRNAs play critical roles in heart disease. In addition to differential miRNA expression, miRNA-mediated control is also affected by variable miRNA processing or alternative 3′-end cleavage and polyadenylation (APA) of their mRNA targets. To what extent these phenomena play a role in the heart remains unclear. We sought to explore miRNA processing and mRNA APA in cardiomyocytes, and whether these change during cardiac hypertrophy. Thoracic aortic constriction (TAC) was performed to induce hypertrophy in C57BL/6J mice. RNA extracted from cardiomyocytes of sham-treated, pre-hypertrophic (2days post-TAC), and hypertrophic (7days post-TAC) mice was subjected to small RNA- and poly(A)-test sequencing (PAT-Seq). Differential expression analysis matched expectations; nevertheless we identified ~400 mRNAs and hundreds of noncoding RNA loci as altered with hypertrophy for the first time. Although multiple processing variants were observed for many miRNAs, there was little change in their relative proportions during hypertrophy. PAT-Seq mapped ~48,000 mRNA 3′-ends, identifying novel 3′ untranslated regions (3′UTRs) for over 7000 genes. Importantly, hypertrophy was associated with marked changes in APA with a net shift from distal to more proximal mRNA 3′-ends, which is predicted to decrease overall miRNA repression strength. We independently validated several examples of 3′UTR proportion change and showed that alternative 3′UTRs associate with differences in mRNA translation. Our work suggests that APA contributes to altered gene expression with the development of cardiomyocyte hypertrophy and provides a rich resource for a systems-level understanding of miRNA-mediated regulation in physiological and pathological states of the heart.

Natural antisense RNA promotes 3' end processing and maturation of MALAT1 lncRNA.

The RNase P-mediated endonucleolytic cleavage plays a crucial role in the 3′ end processing and cellular accumulation of MALAT1, a nuclear-retained long noncoding RNA that promotes malignancy. The regulation of this cleavage event is largely undetermined. Here we characterize a broadly expressed natural antisense transcript at the MALAT1 locus, designated as TALAM1, that positively regulates MALAT1 levels by promoting the 3′ end cleavage and maturation of MALAT1 RNA. TALAM1 RNA preferentially localizes at the site of transcription, and also interacts with MALAT1 RNA. Depletion of TALAM1 leads to defects in the 3′ end cleavage reaction and compromises cellular accumulation of MALAT1. Conversely, overexpression of TALAM1 facilitates the cleavage reaction in trans. Interestingly, TALAM1 is also positively regulated by MALAT1 at the level of both transcription and RNA stability. Together, our data demonstrate a novel feed-forward positive regulatory loop that is established to maintain the high cellular levels of MALAT1, and also unravel the existence of sense-antisense mediated regulatory mechanism for cellular lncRNAs that display RNase P-mediated 3′ end processing.

Diversity of transcripts and transcript processing forms in plastids of the dinoflagellate alga Karenia mikimotoi.

Plastids produce a vast diversity of transcripts. These include mature transcripts containing coding sequences, and their processing precursors, as well as transcripts that lack direct coding functions, such as antisense transcripts. Although plastid transcriptomes have been characterised for many plant species, less is known about the transcripts produced in other plastid lineages. We characterised the transcripts produced in the fucoxanthin-containing plastids of the dinoflagellate alga Karenia mikimotoi. This plastid lineage, acquired through tertiary endosymbiosis, utilises transcript processing pathways that are very different from those found in plants and green algae, including 3′ poly(U) tail addition, and extensive substitutional editing of transcript sequences. We have sequenced the plastid transcriptome of K. mikimotoi, and have detected evidence for divergent evolution of fucoxanthin plastid genomes. We have additionally characterised polycistronic and monocistronic transcripts from two plastid loci, psbD-tRNAMet-ycf4 and rpl36-rps13-rps11. We find evidence for a range of transcripts produced from each locus that differ in terms of editing state, 5′ end cleavage position, and poly(U) tail addition. Finally, we identify antisense transcripts in K. mikimotoi, which appear to undergo different processing events from the corresponding sense transcripts. Overall, our study provides insights into the diversity of transcripts and processing intermediates found in plastid lineages across the eukaryotes.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-015-0408-9) contains supplementary material, which is available to authorized users.

Integrator mediates the biogenesis of enhancer RNAs.

Integrator is a multi-subunit complex stably associated with the C-terminal domain (CTD) of RNA polymerase II (RNAPII) 1. Integrator is endowed with a core catalytic RNA endonuclease activity, which is required for the 3′-end processing of non-polyadenylated RNAPII-dependent uridylate-rich small nuclear RNA genes (UsnRNAs) 1. Here, we examined the requirement of Integrator in the biogenesis of transcripts derived from distal regulatory elements (enhancers) involved in tissue- and temporal-specific regulation of gene expression 2–5. Integrator is recruited to enhancers and super-enhancers in a stimulus-dependent manner. Functional depletion of Integrator subunits diminishes the signal-dependent induction of eRNAs and abrogates the stimulus-induced enhancer-promoter chromatin looping. Global nuclear run-on and RNAPII profiling reveals a role for Integrator in 3′-end cleavage of eRNAs primary transcripts leading to transcriptional termination. In the absence of Integrator, eRNAs remain bound to RNAPII and their primary transcripts accumulates. Importantly, the induction of eRNAs and gene expression responsiveness requires the catalytic activity of Integrator complex. We propose a role for Integrator in biogenesis of eRNAs and enhancer function in metazoans.

Quantification of co-transcriptional splicing from RNA-Seq data

During gene expression, protein-coding transcripts are shaped by multiple processing events: 5′ end capping, pre-mRNA splicing, RNA editing, and 3′ end cleavage and polyadenylation. These events are required to produce mature mRNA, which can be subsequently translated. Nearly all of these RNA processing steps occur during transcription, while the nascent RNA is still attached to the DNA template by RNA polymerase II (i.e. co-transcriptionally). Polyadenylation occurs after 3′ end cleavage or post-transcriptionally. Pre-mRNA splicing – the removal of introns and ligation of exons – can be initiated and concluded co-transcriptionally, although this is not strictly required. Recently, a number of studies using global methods have shown that the majority of splicing is co-transcriptional, yet not all published studies agree in their conclusions. Short read sequencing of RNA (RNA-Seq) is the prevailing approach to measuring splicing levels in nascent RNA, mRNA or total RNA. Here, we compare four different strategies for analyzing and quantifying co-transcriptional splicing. To do so, we reanalyze two nascent RNA-Seq datasets of the same species, but different cell type and RNA isolation procedure. Average co-transcriptional splicing values calculated on a per intron basis are similar, independent of the strategy used. We emphasize the technical requirements for identifying co-transcriptional splicing events with high confidence, e.g. how to calculate co-transcriptional splicing from nascent RNA- versus mRNA-Seq data, the number of biological replicates needed, depletion of polyA+RNA, and appropriate normalization. Finally, we present guidelines for planning a nascent RNA-Seq experiment.

Global Promotion of Alternative Internal Exon Usage by mRNA 3′ End Formation Factors

The mechanisms that regulate alternative precursor mRNA (pre-mRNA) splicing are largely unknown. Here, we perform an RNAi screen to identify factors required for alternative splicing regulation by RBFOX2, an RNA-binding protein that promotes either exon inclusion or exclusion. Unexpectedly, we find that two mRNA 3' end formation factors, cleavage and polyadenylation specificity factor (CPSF) and SYMPK, are RBFOX2 cofactors for both inclusion and exclusion of internal exons. RBFOX2 interacts with CPSF/SYMPK and recruits it to the pre-mRNA. RBFOX2 and CPSF/SYMPK then function together to regulate binding of the early intron recognition factors U2AF and U1 small nuclear ribonucleoprotein particle (snRNP). Genome-wide analysis reveals that CPSF also mediates alternative splicing of many internal exons in the absence of RBFOX2. Accordingly, we show that CPSF/SYMPK is also a cofactor of NOVA2 and heterologous nuclear ribonucleoprotein A1 (HNRNPA1), RNA-binding proteins that also regulate alternative splicing. Collectively, our results reveal an unanticipated role for mRNA 3' end formation factors in global promotion of alternative splicing.

On the occasion of the 20th anniversary of the RNA journal.

On the wonderful occasion of the RNA journal's 20th anniversary, I think one can say without a doubt that the RNA field has been an amazing source of excitement and remarkable advances that have inseminated multiple fields across biology and medicine. The RNA journal reflects the vibrant and collegial RNA community, but the journal's success is also a tribute to the superb leadership, integrity, and scholarship of its Editor, Tim Nilsen, and dedicated Editorial Board. I feel very fortunate to have been working in this exciting field no less because of the many friendships I have made with wonderful colleagues and mostly the truly exceptional individuals that I have had in my laboratory. The journey, so far, has been phenomenal and full of surprises. Looking back, I would not have been able to foresee the course that my laboratory took since the journal's inception. I will briefly describe some highlights that stand out in my mind from our own research and how we got there. I regret that the concise format precludes mentioning the major contributions and influence that others have had on our work—they are many! My laboratory has a long-standing interest in RNA-binding proteins, RNA-protein complexes (RNPs), and their roles in gene regulation and disease. Most gene regulation in complex eukaryotes occurs post-transcriptionally and is mediated by RNA-binding proteins and small noncoding RNAs. To produce mRNA, the primary gene transcripts of the majority of protein coding genes (pre-mRNAs; historically hnRNAs) are extensively processed to remove translation open reading frame-disrupting introns by splicing, and by 5′-end capping and 3′-end cleavage and polyadenylation (CPA). Splicing is mediated by the spliceosome, comprised of non-coding small nuclear RNPs (major: U1, U2, U4, U5, U6; minor: U11, U12, U4atac, U6atac) and protein factors. The majority of pre-mRNAs have multiple introns, each with 5′- and 3′-splice sites (ss) and multiple polyadenylation signals (PASs) that can be utilized in various alternative combinations to produce diverse mRNA and protein isoforms from the same gene. Pre-mRNA processing initiates co-transcriptionally and completes in the nucleus. The mRNAs are then transported to the cytoplasm where they can be translated and are subsequently degraded. Each of these events is regulated in a cell type and cell cycle stage-dependent manner and in response to external cues. All these processes, including recognition of constitutive and alternative pre-mRNA processing signals, depend on RNA-binding proteins (RBPs or RNP proteins).