Exon Junction Complexes Suppress Spurious Splice Sites to Safeguard Transcriptome Integrity.

Productive splicing of human precursor messenger RNAs (pre-mRNAs) requires the correct selection of authentic splice sites (SS) from the large pool of potential SS. Although SS consensus sequence and splicing regulatory proteins are known to influence SS usage, the mechanisms ensuring the effective suppression of cryptic SS are insufficiently explored. Here, we find that many aberrant exonic SS are efficiently silenced by the exon junction complex (EJC), a multi-protein complex that is deposited on spliced mRNA near the exon-exon junction. Upon depletion of EJC proteins, cryptic SS are de-repressed, leading to the mis-splicing of a broad set of mRNAs. Mechanistically, the EJC-mediated recruitment of the splicing regulator RNPS1 inhibits cryptic 5'SS usage, while the deposition of the EJC core directly masks reconstituted 3'SS, thereby precluding transcript disintegration. Thus, the EJC protects the transcriptome of mammalian cells from inadvertent loss of exonic sequences and safeguards the expression of intact, full-length mRNAs.

Exon Junction Complex Shapes the Transcriptome by Repressing Recursive Splicing.

Recursive splicing (RS) starts by defining an "RS-exon," which is then spliced to the preceding exon, thus creating a recursive 5' splice site (RS-5ss). Previous studies focused on cryptic RS-exons, and now we find that the exon junction complex (EJC) represses RS of hundreds of annotated, mainly constitutive RS-exons. The core EJC factors, and the peripheral factors PNN and RNPS1, maintain RS-exon inclusion by repressing spliceosomal assembly on RS-5ss. The EJC also blocks 5ss located near exon-exon junctions, thus repressing inclusion of cryptic microexons. The prevalence of annotated RS-exons is high in deuterostomes, while the cryptic RS-exons are more prevalent in Drosophila, where EJC appears less capable of repressing RS. Notably, incomplete repression of RS also contributes to physiological alternative splicing of several human RS-exons. Finally, haploinsufficiency of the EJC factor Magoh in mice is associated with skipping of RS-exons in the brain, with relevance to the microcephaly phenotype and human diseases.

Genome-wide CRISPR-Cas9 Interrogation of Splicing Networks Reveals a Mechanism for Recognition of Autism-Misregulated Neuronal Microexons.

Alternative splicing is crucial for diverse cellular, developmental, and pathological processes. However, the full networks of factors that control individual splicing events are not known. Here, we describe a CRISPR-based strategy for the genome-wide elucidation of pathways that control splicing and apply it to microexons with important functions in nervous system development and that are commonly misregulated in autism. Approximately 200 genes associated with functionally diverse regulatory layers and enriched in genetic links to autism control neuronal microexons. Remarkably, the widely expressed RNA binding proteins Srsf11 and Rnps1 directly, preferentially, and frequently co-activate these microexons. These factors form critical interactions with the neuronal splicing regulator Srrm4 and a bi-partite intronic splicing enhancer element to promote spliceosome formation. Our study thus presents a versatile system for the identification of entire splicing regulatory pathways and further reveals a common mechanism for the definition of neuronal microexons that is disrupted in autism.

Spatial Organization of Single mRNPs at Different Stages of the Gene Expression Pathway.

mRNAs form ribonucleoprotein complexes (mRNPs) by association with proteins that are crucial for mRNA metabolism. While the mRNP proteome has been well characterized, little is known about mRNP organization. Using a single-molecule approach, we show that mRNA conformation changes depending on its cellular localization and translational state. Compared to nuclear mRNPs and lncRNPs, association with ribosomes decompacts individual mRNAs, while pharmacologically dissociating ribosomes or sequestering them into stress granules leads to increased compaction. Moreover, translating mRNAs rarely show co-localized 5' and 3' ends, indicating either that mRNAs are not translated in a closed-loop configuration, or that mRNA circularization is transient, suggesting that a stable closed-loop conformation is not a universal state for all translating mRNAs.

Structure and function of pre-mRNA 5'-end capping quality control and 3'-end processing.

Messenger RNA precursors (pre-mRNAs) are produced as the nascent transcripts of RNA polymerase II (Pol II) in eukaryotes and must undergo extensive maturational processing, including 5'-end capping, splicing, and 3'-end cleavage and polyadenylation. This review will summarize the structural and functional information reported over the past few years on the large machinery required for the 3'-end processing of most pre-mRNAs, as well as the distinct machinery for the 3'-end processing of replication-dependent histone pre-mRNAs, which have provided great insights into the proteins and their subcomplexes in these machineries. Structural and biochemical studies have also led to the identification of a new class of enzymes (the DXO family enzymes) with activity toward intermediates of the 5'-end capping pathway. Functional studies demonstrate that these enzymes are part of a novel quality surveillance mechanism for pre-mRNA 5'-end capping. Incompletely capped pre-mRNAs are produced in yeast and human cells, in contrast to the general belief in the field that capping always proceeds to completion, and incomplete capping leads to defects in splicing and 3'-end cleavage in human cells. The DXO family enzymes are required for the detection and degradation of these defective RNAs.

Synthetic pre-microRNAs reveal dual-strand activity of miR-34a on TNF-α.

Functional microRNAs (miRNAs) are produced from both arms of their precursors (pre-miRNAs). Their abundances vary in context-dependent fashion spatiotemporarily and there is mounting evidence of regulatory interplay between them. Here, we introduce chemically synthesized pre-miRNAs (syn-pre-miRNAs) as a general class of accessible, easily transfectable mimics of pre-miRNAs. These are RNA hairpins, identical in sequence to natural pre-miRNAs. They differ from commercially available miRNA mimics through their complete hairpin structure, including any regulatory elements in their terminal-loop regions and their potential to introduce both strands into RISC. They are distinguished from transcribed pre-miRNAs by their terminal 5' hydroxyl groups and their precisely defined terminal nucleotides. We demonstrate with several examples how they fully recapitulate the properties of pre-miRNAs, including their processing by Dicer into functionally active 5p; and 3p-derived mature miRNAs. We use syn-pre-miRNAs to show that miR-34a uses its 5p and 3p miRNAs in two pathways: apoptosis during TGF-ß signaling, where SIRT1 and SP4 are suppressed by miR-34a-5p and miR-34a-3p, respectively; and the lipopolysaccharide (LPS)-activation of primary human monocyte-derived macrophages, where TNF (TNFα) is suppressed by miR-34a-5p indirectly and miR-34a-3p directly. Our results add to growing evidence that the use of both arms of a miRNA may be a widely used mechanism. We further suggest that syn-pre-miRNAs are ideal and affordable tools to investigate these mechanisms.

Deciphering 3'ss selection in the yeast genome reveals an RNA thermosensor that mediates alternative splicing.

Poor understanding of the spliceosomal mechanisms to select intronic 3' ends (3'ss) is a major obstacle to deciphering eukaryotic genomes. Here, we discern the rules for global 3'ss selection in yeast. We show that, in contrast to the uniformity of yeast splicing, the spliceosome uses all available 3'ss within a distance window from the intronic branch site (BS), and that in ∼70% of all possible 3'ss this is likely to be mediated by pre-mRNA structures. Our results reveal that one of these RNA folds acts as an RNA thermosensor, modulating alternative splicing in response to heat shock by controlling alternate 3'ss availability. Thus, our data point to a deeper role for the pre-mRNA in the control of its own fate, and to a simple mechanism for some alternative splicing.

The role of the precursor structure in the biogenesis of microRNA.

The human genome contains more than 1,000 microRNA (miRNA) genes, which are transcribed mainly by RNA polymerase II. The canonical pathway of miRNA biogenesis includes the nuclear processing of primary transcripts (pri-miRNAs) by the ribonuclease Drosha and further cytoplasmic processing of pre-miRNAs by the ribonuclease Dicer. This review discusses the issue of miRNA end heterogeneity generated primarily by Drosha and Dicer cleavage and focuses on the structural aspects of the Dicer step of miRNA biogenesis. We examine the structures of miRNA precursors, both predicted and experimentally determined, as well as the influence of various motifs that disturb the regularity of pre-miRNA structure on Dicer cleavage specificity. We evaluate the structural determinants of the length diversity of miRNA generated by Dicer from different precursors and highlight the importance of asymmetrical motifs. Finally, we discuss the impact of Dicer protein partners on cleavage efficiency and specificity and propose the contribution of pre-miRNA structural plasticity to the dynamics of the dicing complex.

PRP-17 and the pre-mRNA splicing pathway are preferentially required for the proliferation versus meiotic development decision and germline sex determination in Caenorhabditis elegans.

In C. elegans, the decision between germline stem cell proliferation and entry into meiosis is controlled by GLP-1 Notch signaling, which promotes proliferation through repression of the redundant GLD-1 and GLD-2 pathways that direct meiotic entry. We identify prp-17 as another gene functioning downstream of GLP-1 signaling that promotes meiotic entry, largely by acting on the GLD-1 pathway, and that also functions in female germline sex determination. PRP-17 is orthologous to the yeast and human pre-mRNA splicing factor PRP17/CDC40 and can rescue the temperature-sensitive lethality of yeast PRP17. This link to splicing led to an RNAi screen of predicted C. elegans splicing factors in sensitized genetic backgrounds. We found that many genes throughout the splicing cascade function in the proliferation/meiotic entry decision and germline sex determination indicating that splicing per se, rather than a novel function of a subset of splicing factors, is necessary for these processes.

KSRP promotes the maturation of a group of miRNA precursors.

microRNAs (miRNAs) are small noncodingRNAs that down-regulate gene expression by reducing stability and/or translation of target mRNAs. In animals, miRNAs arise from sequential processing of hairpin primary transcripts by two RNAse III domain-containing enzymes, namely Drosha and Dicer, to generate a mature form of about 22 nucleotides. In this chapter we discuss our latest findings indicating that KSRP is an integral component of both Drosha and Dicer complexes. KSRP binds to the terminal loop sequence of a subset of miRNA precursors promoting their maturation. Our data indicate that the terminal loop is a pivotal structure where activators of miRNA processing as well as repressors of miRNA processing act in a coordinated way to convert cellular signals into changes in miRNA expression processing. This uncovers a new level of complexity of miRNA mechanisms for gene expression regulation.

Regulation of pre-miRNA processing.

microRNAs are endogenously expressed 21 nucleotide noncoding RNAs. microRNA-mediated regulation of the translation of specific mRNA is implicated in a range of developmental processes and pathologies. As such, miRNA expression is tightly controlled in normal development by both transcriptional and post-transcriptional mechanisms. This chapter is concerned with the control of pre-miRNA processing of individual miRNAs by specific factors. It is focussed on the regulation of a subset of miRNAs by the RNA-binding protein Lin28/LIN-28. We discuss how Lin28/LIN-28 can sequester pre-let-7 miRNA precursor to prevent Dicer-mediated processing. We describe how interaction of pre-let-7 with Lin28/ LIN-28 leads to pre-let-7 uridylation and subsequent degradation. Finally, we analyze how let-7 and Lin28/LIN-28 together act as a highly conserved developmental switch that controls stem cell differentiation in C. elegans and mammals.

In vivo selection of kinase-responsive RNA elements controlling alternative splicing.

Alternative pre-mRNA splicing is often controlled by cell signals, for example, those activating the cAMP-dependent protein kinase (PKA) or the Ca2+/calmodulin-dependent protein kinase IV (CaMKIV). We have shown that CaMKIV regulates alternative splicing through short CA repeats and hnRNP L. Here we use a splicing reporter that shows PKA/CaMKIV promotion of exon inclusion to select from exons containing random 13-nt sequences for RNA elements responsive to the kinases in cultured cells. This selection not only identified both PKA- and CaMKIV-responsive elements that are similar to the CaMKIV-responsive RNA element 1 (CaRRE1) or CA repeats, but also A-rich elements not previously known to respond to these kinases. Consistently, hnRNP L is identified as a factor binding the CA-rich elements. Analyses of the motifs in the highly responsive elements indicate that they are indeed critical for the kinase effect and are enriched in alternative exons. Interestingly, a CAAAAAA motif is sufficient for the PKA/CaMKIV-regulated splicing of the exon 16 of the CaMK kinase beta1 (CaMKK2) transcripts, implying a role of this motif in signaling cross-talk or feedback regulation between these kinases through alternative splicing. Therefore, these experiments identified a group of RNA elements responsive to PKA and CaMKIV from in vivo selection. This also provides an approach for selecting RNA elements similarly responsive to other cell signals controlling alternative splicing.

Alternative splicing and disease.

Splicing is a molecular mechanism, by which introns are removed from an mRNA precursor and exons are ligated to form a mature mRNA. Mutations that cause defects in the splicing mechanism are known to be responsible for many diseases, including cystic fibrosis and familial dysautonomia. If mutations that cause defects in splicing are responsible for such severe deleterious phenotypic differences, it is possible that mutations in splicing are also responsible for mildly deleterious phenotypic differences. Although deleterious mutations are rapidly eliminated from the population by purifying selection, the selection against mild deleterious effects is not as strong. Since mildly deleterious mutations have a chance of surviving natural selection, we might be mistakenly referring to these mutations as neutral variation between individuals. Splicing has also been shown to be seriously affected in cancer. Examination of cancerous tissues revealed alterations in expression levels of genes involved in mRNA processing and also a slight reduction in the level of exon skipping--the most common form of alternative splicing in humans. This implies that defects in genes involved in the regulation of splicing in cancerous tissues affect the delicate regulation of the inclusion level of alternatively skipped exons, shifting their mode of splicing back to constitutive. It may be that splicing silencers play a more prominent role in alternative splicing regulation than previously anticipated.

Identification of cis-acting elements involved in acetylcholinesterase RNA alternative splicing.

The 3' end of Acetylcholinesterase (AChE) pre-mRNA is processed by a complex mechanism of alternative splicing. Three different transcripts are generated and called R, H and T according respectively to the intron (intron 4') or exons (5 or 6) retained in the mature RNA. The relative expression of the specific transcripts depends on cell type, developmental stage or pathophysiological conditions. The aim of our study was to identify sequences involved in AChE pre-mRNA splicing choices. For this purpose, we constructed a minigene in which the constitutive exons were fused and followed by the entire alternative domain without 3' UTR. We transfected the wild-type or minigene mutated in the alternative domain in muscle or COS-7 cells and identified the splicing products by RPA, RT-PCR and sedimentation coefficients of the enzymatic molecular forms. We find that the alternative splicing domain contains most of the necessary signals to control splicing choices in skeletal muscle cells with the coding sequences of the domain having little effect on the splicing outcome. A branch point at an unusual location 278 nt from the 3' acceptor site of exon 6 is characterized. We further identify several regulatory sequences in the non-coding sequence of exon 5 that regulate the splicing pattern. Sequences that control the splice to exon 5 and those which influence intron 4' retention or splicing to exon 6 appear to be distinct.

Ligand-induced sequestering of branchpoint sequence allows conditional control of splicing.

BACKGROUND: Despite tremendous progress in understanding the mechanisms of constitutive and alternative splicing, an important and widespread step along the gene expression pathway, our ability to deliberately regulate gene expression at this step remains rudimentary. The present study was performed to investigate whether a theophylline-dependent "splice switch" that sequesters the branchpoint sequence (BPS) within RNA-theophylline complex can regulate alternative splicing. RESULTS: We constructed a series of pre-mRNAs in which the BPS was inserted within theophylline aptamer. We show that theophylline-induced sequestering of BPS inhibits pre-mRNA splicing both in vitro and in vivo in a dose-dependent manner. Several lines of evidence suggest that theophylline-dependent inhibition of splicing is highly specific, and thermodynamic stability of RNA-theophylline complex as well as the location of BPS within this complex affects the efficiency of splicing inhibition. Finally, we have constructed an alternative splicing model pre-mRNA substrate in which theophylline caused exon skipping both in vitro and in vivo, suggesting that a small molecule-RNA interaction can modulate alternative splicing. CONCLUSION: These findings provide the ability to control splicing pattern at will and should have important implications for basic, biotechnological, and biomedical research.

Control of alternative pre-mRNA splicing by Ca(++) signals.

Alternative pre-mRNA splicing is a common way of gene expression regulation in metazoans. The selective use of specific exons can be modulated in response to various manipulations that alter Ca(++) signals, particularly in neurons. A number of splicing factors have also been found to be controlled by Ca(++) signals. Moreover, pre-mRNA elements have been identified that are essential and sufficient to mediate Ca(++)-regulated splicing, providing model systems for dissecting the involved molecular components. In neurons, this regulation likely contributes to the fine-tuning of neuronal properties.

Dual role for argonautes in microRNA processing and posttranscriptional regulation of microRNA expression.

MicroRNAs are small endogenous noncoding RNAs involved in posttranscriptional gene regulation. During microRNA biogenesis, Drosha and Dicer process the primary transcript (pri-miRNA) through a precursor hairpin (pre-miRNA) to the mature miRNA. The miRNA is incorporated into the RNA-Induced Silencing Complex (RISC) with Argonaute proteins, the effector molecules in RNA interference (RNAi). Here, we show that all Argonautes elevate mature miRNA expression posttranscriptionally, independent of RNase activity. Also, we identify a role for the RISC slicer Argonaute2 (Ago2) in cleaving the pre-miRNA to an additional processing intermediate, termed Ago2-cleaved precursor miRNA or ac-pre-miRNA. This endogenous, on-pathway intermediate results from cleavage of the pre-miRNA hairpin 12 nucleotides from its 3'-end. By analogy to siRNA processing, Ago2 cleavage may facilitate removal of the nicked passenger strand from RISC after maturation. The multiple roles of Argonautes in the RNAi effector phase and miRNA biogenesis and maturation suggest coordinate regulation of microRNA expression and function.

The Hsp40 chaperone Jjj1 is required for the nucleo-cytoplasmic recycling of preribosomal factors in Saccharomyces cerevisiae.

Ribosome biogenesis is a major conserved cellular pathway that requires both ribosomal proteins and many preribosomal factors. Most of the pre-60S factors are recycled into the nucleus; some of them shuttle between the nucleus and the cytoplasm while a few others, like Rei1, are strictly cytoplasmic and are mostly involved in the dissociation/recycling of the pre-60S shuttling factors. Here, we investigated the role of the Jjj1 Hsp40 chaperone in ribosome biogenesis. The absence of Jjj1 leads to a cold sensitive phenotype, a defect in the relative amount of the large ribosomal subunit with the appearance of halfmers, and to cytoplasmic accumulation of shuttling factors such as Arx1 and Alb1, which stay bound to the pre-60S particles. Jjj1 is, thus, a novel pre-60S factor involved in the last cytoplasmic steps of the large ribosomal subunit biogenesis. We report the biochemical association of Jjj1 and Rei1 to similar pre-60S complexes, their two-hybrid interactions, and their functional links. Altogether, these results indicate that Rei1 and Jjj1 share many common features. However, while the functions of Jjj1 and Rei1 partially overlap, we could distinguish specific role of the two proteins in Arx1/Alb1 and Tif6 recycling. We propose that Jjj1 is preferentially required for the release of Arx1 and Alb1 shuttling factors from the cytoplasmic pre-60S particles while Rei1 is preferentially involved in their recycling.

The tripartite motif of nuclear factor 7 is required for its association with transcriptional units.

In amphibian oocytes, the maternal nuclear factor NF7 associates with the elongating pre-mRNAs present on the numerous lateral loops of the lampbrush chromosomes. Here, we have purified NF7 from an oocyte extract by using a combination of ion-exchange chromatography and gel filtration chromatography and demonstrated for the first time that nucleoplasmic NF7 exists primarily as free homotrimers. We confirmed the in vivo homotrimerization of NF7 by using a glutaraldehyde cross-linking assay, and we further showed that it only requires the coiled-coil domain of the NF7 tripartite motif/RBCC motif. Interestingly, we also obtained evidence that NF7 is recruited to the nucleus as a homotrimer, and expression of several mutated forms of NF7 in oocytes demonstrated that both the coiled coil and B box of NF7 are required for its chromosomal association. Together, these data strongly suggest that the interaction of NF7 with the active transcriptional units of RNA polymerase II is mediated by a trimeric B box. Finally, and in agreement with a role for NF7 in pre-mRNA maturation, we obtained evidence supporting the idea that NF7 associates with Cajal bodies.