Alternative exon definition events control the choice between nuclear retention and cytoplasmic export of U11/U12-65K mRNA

Cellular homeostasis of the minor spliceosome is regulated by a negative feed-back loop that targets U11-48K and U11/U12-65K mRNAs encoding essential components of the U12-type intron-specific U11/U12 di-snRNP. This involves interaction of the U11 snRNP with an evolutionarily conserved splicing enhancer giving rise to unproductive mRNA isoforms. In the case of U11/U12-65K, this mechanism controls the length of the 3' untranslated region (3'UTR). We show that this process is dynamically regulated in developing neurons and some other cell types, and involves a binary switch between translation-competent mRNAs with a short 3'UTR to non-productive isoforms with a long 3'UTR that are retained in the nucleus or/and spliced to the downstream amylase locus. Importantly, the choice between these alternatives is determined by alternative terminal exon definition events regulated by conserved U12-and U2-type 50 splice sites as well as sequence signals used for pre-mRNA cleavage and polyadenylation. We additionally show that U11 snRNP binding to the U11/U12-65K mRNA species with a long 3'UTR is required for their nuclear retention. Together, our studies uncover an intricate molecular circuitry regulating the abundance of a key spliceosomal protein and shed new light on the mechanisms limiting the export of non-productively spliced mRNAs from the nucleus to the cytoplasm.Peer reviewe

Loss of muscleblind splicing factor shortens Caenorhabditis elegans lifespan by reducing the activity of p38 MAPK/PMK-1 and transcription factors ATF-7 and Nrf/SKN-1

Muscleblind-like splicing regulators (MBNLs) are RNA-binding factors that have an important role in developmental processes. Dysfunction of these factors is a key contributor of different neuromuscular degenerative disorders, including Myotonic Dystrophy type 1 (DM1). Since DM1 is a multisystemic disease characterized by symptoms resembling accelerated aging, we asked which cellular processes do MBNLs regulate that make them necessary for normal lifespan. By utilizing the model organism Caenorhabditis elegans, we found that loss of MBL-1 (the sole ortholog of mammalian MBNLs), which is known to be required for normal lifespan, shortens lifespan by decreasing the activity of p38 MAPK/PM K-1 as well as the function of transcription factors ATF-7 and SKN-1. Furthermore, we show that mitochondrial stress caused by the knockdown of mitochondrial electron transport chain components promotes the longevity of mbl-1 mutants in a partially PMK-1-dependent manner. Together, the data establish a mechanism of how DM1-associated loss of muscleblind affects lifespan. Furthermore, this study suggests that mitochondrial stress could alleviate symptoms caused by the dysfunction of muscleblind splicing factor, creating a potential approach to investigate for therapy.Peer reviewe

Chromosomal instability by mutations in the novel minor spliceosome component CENATAC

Aneuploidy is the leading cause of miscarriage and congenital birth defects, and a hallmark of cancer. Despite this strong association with human disease, the genetic causes of aneuploidy remain largely unknown. Through exome sequencing of patients with constitutional mosaic aneuploidy, we identified biallelic truncating mutations in CENATAC (CCDC84). We show that CENATAC is a novel component of the minor (U12-dependent) spliceosome that promotes splicing of a specific, rare minor intron subtype. This subtype is characterized by AT-AN splice sites and relatively high basal levels of intron retention. CENATAC depletion or expression of disease mutants resulted in excessive retention of AT-AN minor introns in similar to 100 genes enriched for nucleocytoplasmic transport and cell cycle regulators, and caused chromosome segregation errors. Our findings reveal selectivity in minor intron splicing and suggest a link between minor spliceosome defects and constitutional aneuploidy in humans.Peer reviewe

The Muscleblind-like protein MBL-1 regulates microRNA expression in Caenorhabditis elegans through an evolutionarily conserved autoregulatory mechanism.

The Muscleblind-like (MBNL) family is a highly conserved set of RNA-binding proteins (RBPs) that regulate RNA metabolism during the differentiation of various animal tissues. Functional insufficiency of MBNL affects muscle and central nervous system development, and contributes to the myotonic dystrophies (DM), a set of incurable multisystemic disorders. Studies on the regulation of MBNL genes are essential to provide insight into the gene regulatory networks controlled by MBNL proteins and to understand how dysregulation within these networks causes disease. In this study, we demonstrate the evolutionary conservation of an autoregulatory mechanism that governs the function of MBNL proteins by generating two distinct protein isoform types through alternative splicing. Our aim was to further our understanding of the regulatory principles that underlie this conserved feedback loop in a whole-organismal context, and to address the biological significance of the respective isoforms. Using an alternative splicing reporter, our studies show that, during development of the Caenorhabditis elegans central nervous system, the orthologous mbl-1 gene shifts production from long protein isoforms that localize to the nucleus to short isoforms that also localize to the cytoplasm. Using isoform-specific CRISPR/Cas9-generated strains, we showed that expression of short MBL-1 protein isoforms is required for healthy neuromuscular function and neurodevelopment, while expression of long MBL-1 protein isoforms is dispensable, emphasizing a key role for cytoplasmic functionalities of the MBL-1 protein. Furthermore, RNA-seq and lifespan analyses indicated that short MBL-1 isoforms are crucial regulators of miRNA expression and, in consequence, required for normal lifespan. In conclusion, this study provides support for the disruption of cytoplasmic RNA metabolism as a contributor in myotonic dystrophy and paves the way for further exploration of miRNA regulation through MBNL proteins during development and in disease models

Evolutionarily conserved exon definition interactions with U11 snRNP mediate alternative splicing regulation on U11–48K and U11/U12–65K genes

<div><p>Many splicing regulators bind to their own pre-mRNAs to induce alternative splicing that leads to formation of unstable mRNA isoforms. This provides an autoregulatory feedback mechanism that regulates the cellular homeostasis of these factors. We have described such an autoregulatory mechanism for two core protein components, U11–48K and U11/U12–65K, of the U12-dependent spliceosome. This regulatory system uses an atypical splicing enhancer element termed USSE (U11 snRNP-binding splicing enhancer), which contains two U12-type consensus 5′ splice sites (5′ss). Evolutionary analysis of the USSE element from a large number of animal and plant species indicate that USSE sequence must be located 25–50 nt downstream from the target 3′ splice site (3′ss). Together with functional evidence showing a loss of USSE activity when this distance is reduced and a requirement for RS-domain of U11–35K protein for 3′ss activation, our data suggests that U11 snRNP bound to USSE uses exon definition interactions for regulating alternative splicing. However, unlike standard exon definition where the 5′ss bound by U1 or U11 will be subsequently activated for splicing, the USSE element functions similarly as an exonic splicing enhancer and is involved only in upstream splice site activation but does not function as a splicing donor. Additionally, our evolutionary and functional data suggests that the function of the 5′ss duplication within the USSE elements is to allow binding of two U11/U12 di-snRNPs that stabilize each others' binding through putative mutual interactions.</p></div

<i>U11/U12-65K</i> transcripts with a long 3′UTR are associated with transcriptional read-through.

<p><b>(A)</b> Following transfection with mock or block morpholinos in HEK293 cells, RT-PCR was performed to detect both <i>65K</i> isoforms simultaneously, using a reverse primer binding at the canonical terminal exon (upper panel), or located in the IE (lower panel). Amplicons were separated on a 1% agarose gel. Endoporter transfection reagent was used for the (-) control sample. The marker (M) is Generuler (Thermo Scientific: #SM0331). <b>(B)</b> qRT-PCR analysis of IE expression level for morpholino transfected samples normalized against total <i>65K</i> expression. Error bars represent the standard deviation of 3 biological replicates and the asterisk indicates p-value < 0.05 in a two-tailed Student′s t-test in (B), (D), (F) and (G). <b>(C)</b> Upper panel. Schematics of the reporter plasmid construction. Shaded boxes depict <i>65K</i> sequences cloned into pGL4.13. Lower panel indicate mutations of the putative poly(A) sites (in bold and underlined) of the <i>65K</i> gene to generate the pAdel reporter construct. <b>(D)</b> CHO cells were transfected with wt and pAdel reporter constructs and construct <i>65K</i> long-3′UTR and short-3′UTR splicing isoforms were quantified by qPCR. Fold change is relative to the wt construct <i>65K</i> short-3′UTR splicing isoform. <b>(E)</b> Multiplex (upper panel) and normal RT-PCR of the samples analyzed in (D) were performed and amplicons separated on 1.3% agarose gel. Primers (shown as arrows) were designed to quantify construct <i>65K</i> 3′UTR splicing isoform ratio (upper panel) and read-through and IE splicing of construct <i>65K</i> short- and long-3′UTR splicing isoform transcripts. Dashed lines indicate an exon-spanning primer and an asterisk a construct-specific isoform (splicing event could not be found from endogenous <i>65K</i> gene) skipping the short-3′UTR specific exon and directly splicing towards to IE. <b>(F)</b> qPCR quantification of construct <i>65K</i> 3′UTR splicing isoform transcripts that undergo IE splicing from samples analyzed in (E). <b>(G)</b> RT-PCR analysis of expression of short- and long-3′UTR splicing isoform transcripts that read-through past the poly(A) site but do not splice to the IE (RT transcripts) from samples analyzed in (E). Fold change is relative to the wt RT transcript expression and samples were normalized against total construct <i>65K</i> short- or long-3′UTR splicing isoform levels.</p

Stable nuclear retention of the <i>65K</i> long-3′UTR splicing isoform.

<p><b>(A)</b> A schematics showing the location of Cy5-labeled smFISH probes. LONG set: probes bind specifically long-3′UTR isoform, TOTAL set: probes bind both long and short-3′UTR isoform. <b>(B)</b> smFISH for mock morpholino treated HEK293 cells with TOTAL probe set and in <b>(C)</b> LONG set. <b>(D)</b> smFISH for USSE block morpholino treated cells with TOTAL set and in <b>(E)</b> LONG set. <b>(F)</b> Spots were counted for B (n = 811)), C (n = 346), D (n = 545), and E (n = 140) in the nucleus and cytoplasm using the FISH-QUANT software [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006824#pgen.1006824.ref097" target="_blank">97</a>]. Asterisks indicate two-tailed p-value < 0.0001 in Fisher′s test. <b>(G)</b> Following cellular fractionation of HEK293 cells, multiplex RT-PCR was performed to measure levels of <i>65K</i> long and short-3′UTR isoforms (T: total fraction, C: cytoplasmic fraction, N: nuclear fraction). Black arrows show primer location: forward primer targets both isoforms, while reverse primers target either long or short-3′UTR isoform. <b>(H)</b> Western Blot using antibodies against GAPDH and H3 as quality control for cellular fractionation performed in panel G (T: total fraction, C: cytoplasmic fraction, N: nuclear fraction). <b>(I)</b>. HEK293 cells were treated with DRB and mRNA levels were assayed at regular time intervals by qRT-PCR for the two <i>65K</i> mRNA isoforms and <i>TSP1</i> mRNA as indicated. U6 snRNA expression was used for normalization. Error bars represent standard deviation for three biological replicates.</p