U1A is a positive regulator of the expression of heterologous and cellular genes involved in cell proliferation and migration

Here, we show that direct recruitment of U1A to target transcripts can increase gene expression. This is a new regulatory role, in addition to previous knowledge showing that U1A decreases the levels of U1A mRNA and other specific targets. In fact, genome-wide, U1A more often increases rather than represses gene expression and many U1A-upregulated transcripts are directly bound by U1A according to individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP) studies. Interestingly, U1A-mediated positive regulation can be transferred to a heterologous system for biotechnological purposes. Finally, U1A-bound genes are enriched for those involved in cell cycle and adhesion. In agreement with this, higher U1A mRNA expression associates with lower disease-free survival and overall survival in many cancer types, and U1A mRNA levels positively correlate with those of some oncogenes involved in cell proliferation. Accordingly, U1A depletion leads to decreased expression of these genes and the migration-related gene CCN2/CTGF, which shows the strongest regulation by U1A. A decrease in U1A causes a strong drop in CCN2 expression and CTGF secretion and defects in the expression of CTGF EMT targets, cell migration, and proliferation. These results support U1A as a putative therapeutic target for cancer treatment. In addition, U1A-binding sequences should be considered in biotechnological applications

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

Increased in vivo inhibition of gene expression by combining RNA interference and U1 inhibition

Inhibition of gene expression can be achieved with RNA interference (RNAi) or U1 small nuclear RNA—snRNA—interference (U1i). U1i is based on U1 inhibitors (U1in), U1 snRNA molecules modified to inhibit polyadenylation of a target pre-mRNA. In culture, we have shown that the combination of RNAi and U1i results in stronger inhibition of reporter or endogenous genes than that obtained using either of the techniques alone. We have now used these techniques to inhibit gene expression in mice. We show that U1ins can induce strong inhibition of the expression of target genes in vivo. Furthermore, combining U1i and RNAi results in synergistic inhibitions also in mice. This is shown for the inhibition of hepatitis B virus (HBV) sequences or endogenous Notch1. Surprisingly, inhibition obtained by combining a U1in and a RNAi mediator is higher than that obtained by combining two U1ins or two RNAi mediators. Our results suggest that RNAi and U1i cooperate by unknown mechanisms to result in synergistic inhibitions. Analysis of toxicity and specificity indicates that expression of U1i inhibitors is safe. Therefore, we believe that the combination of RNAi and U1i will be a good option to block damaging endogenous genes, HBV and other infectious agents in vivo

Lessons from non-canonical splicing

Recent improvements in experimental and computational techniques that are used to study the transcriptome have enabled an unprecedented view of RNA processing, revealing many previously unknown non-canonical splicing events. This includes cryptic events located far from the currently annotated exons and unconventional splicing mechanisms that have important roles in regulating gene expression. These non-canonical splicing events are a major source of newly emerging transcripts during evolution, especially when they involve sequences derived from transposable elements. They are therefore under precise regulation and quality control, which minimizes their potential to disrupt gene expression. We explain how non-canonical splicing can lead to aberrant transcripts that cause many diseases, and also how it can be exploited for new therapeutic strategies

Increased in vivo inhibition of gene expression by combining RNA interference and U1 inhibition

Inhibition of gene expression can be achieved with RNA interference (RNAi) or U1 small nuclear RNA-snRNA-interference (U1i). U1i is based on U1 inhibitors (U1in), U1 snRNA molecules modified to inhibit polyadenylation of a target pre-mRNA. In culture, we have shown that the combination of RNAi and U1i results in stronger inhibition of reporter or endogenous genes than that obtained using either of the techniques alone. We have now used these techniques to inhibit gene expression in mice. We show that U1ins can induce strong inhibition of the expression of target genes in vivo. Furthermore, combining U1i and RNAi results in synergistic inhibitions also in mice. This is shown for the inhibition of hepatitis B virus (HBV) sequences or endogenous Notch1. Surprisingly, inhibition obtained by combining a U1in and a RNAi mediator is higher than that obtained by combining two U1ins or two RNAi mediators. Our results suggest that RNAi and U1i cooperate by unknown mechanisms to result in synergistic inhibitions. Analysis of toxicity and specificity indicates that expression of U1i inhibitors is safe. Therefore, we believe that the combination of RNAi and U1i will be a good option to block damaging endogenous genes, HBV and other infectious agents in vivo

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 derepressed, 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

eIF4A3 regulates the TFEB-mediated transcriptional response via GSK3B to control autophagy

During autophagy, the coordinated actions of autophagosomes and lysosomes result in the controlled removal of damaged intracellular organelles and superfluous substrates. The evolutionary conservation of this process and its requirement for maintaining cellular homeostasis emphasizes the need to better dissect the pathways governing its molecular regulation. In our previously performed high-content screen, we assessed the effect of 1530 RNA-binding proteins on autophagy. Among the top regulators, we identified the eukaryotic translation initiation factor 4A-3 (eIF4A3). Here we show that depletion of eIF4A3 leads to a potent increase in autophagosome and lysosome biogenesis and an enhanced autophagic flux. This is mediated by the key autophagy transcription factor, TFEB, which becomes dephosphorylated and translocates from the cytoplasm to the nucleus where it elicits an integrated transcriptional response. We further identified an exon-skipping event in the transcript encoding for the direct TFEB kinase, GSK3B, which leads to a reduction in GSK3B expression and activity. Through analysis of TCGA data, we found a significant upregulation of eIF4A3 expression across several cancer types and confirmed the potential relevance of this newly identified signaling axis in human tumors. Hence, our data suggest a previously unrecognized role for eIF4A3 as a gatekeeper of autophagy through the control of TFEB activation, revealing a new mechanism for autophagy regulation

Recursive splicing in long vertebrate genes

It is generally believed that splicing removes introns as single units from precursor messenger RNA transcripts. However, some long Drosophila melanogaster introns contain a cryptic site, known as a recursive splice site (RS-site), that enables a multi-step process of intron removal termed recursive splicing. The extent to which recursive splicing occurs in other species and its mechanistic basis have not been examined. Here we identify highly conserved RS-sites in genes expressed in the mammalian brain that encode proteins functioning in neuronal development. Moreover, the RS-sites are found in some of the longest introns across vertebrates. We find that vertebrate recursive splicing requires initial definition of an RS-exon that follows the RS-site. The RS-exon is then excluded from the dominant mRNA isoform owing to competition with a reconstituted 5 splice site formed at the RS-site after the first splicing step. Conversely, the RS-exon is included when preceded by cryptic promoters or exons that fail to reconstitute an efficient 5 splice site. Most RS-exons contain a premature stop codon such that their inclusion can decrease mRNA stability. Thus, by establishing a binary splicing switch, RS-sites demarcate different mRNA isoforms emerging from long genes by coupling cryptic elements with inclusion of RS-exons