SR protein-mediated inhibition of CFTR exon 9 inclusion: molecular characterization of the intronic splicing silencer

Buratti, Emanuele; Stuani, Cristiana; Prato, Greta De; Baralle, Francisco E.

doi:10.1093/nar/gkm444

Cited by 47 publications

(50 citation statements)

References 75 publications

(56 reference statements)

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“…Electroporation was used to transfect the CFTR minigene construct (Buratti et al , 2007) into MEFs of the different Tardbp genotypes. Briefly, an Amaxa system (Lonza) was used following manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Mice with endogenous TDP ‐43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis

et al. 2018

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TDP‐43 (encoded by the gene TARDBP) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how TARDBP mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous Tardbp to dissect TDP‐43 function at physiological levels both in vitro and in vivo. Interestingly, we find that mutations within the C‐terminal domain of TDP‐43 lead to a gain of splicing function. Using two different strains, we are able to separate TDP‐43 loss‐ and gain‐of‐function effects. TDP‐43 gain‐of‐function effects in these mice reveal a novel category of splicing events controlled by TDP‐43, referred to as “skiptic” exons, in which skipping of constitutive exons causes changes in gene expression. In vivo, this gain‐of‐function mutation in endogenous Tardbp causes an adult‐onset neuromuscular phenotype accompanied by motor neuron loss and neurodegenerative changes. Furthermore, we have validated the splicing gain‐of‐function and skiptic exons in ALS patient‐derived cells. Our findings provide a novel pathogenic mechanism and highlight how TDP‐43 gain of function and loss of function affect RNA processing differently, suggesting they may act at different disease stages.

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Section: Methodsmentioning

confidence: 99%

Mice with endogenous TDP ‐43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis

et al. 2018

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“…Analogous to the common overlap between ESSs and ISEs, some SR proteins can promote splicing when bound to sites (ESEs) in exons, and also inhibit splicing when bound to intronic (ISS) sites (Kanopka et al 1996;Ibrahim el et al 2005;Buratti et al 2007). Results of a FAS-based screen for ISSs suggest that overlap between ISS and ESE activity could be a general phenomenon (Z. Wang, J. Zhang, X. Xiao, and C.B.…”

Section: Context Dependence Of Sresmentioning

confidence: 99%

Splicing regulation: From a parts list of regulatory elements to an integrated splicing code

Wang¹,

Burge²

2008

RNA

881

761

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Alternative splicing of pre-mRNAs is a major contributor to both proteomic diversity and control of gene expression levels. Splicing is tightly regulated in different tissues and developmental stages, and its disruption can lead to a wide range of human diseases. An important long-term goal in the splicing field is to determine a set of rules or ''code'' for splicing that will enable prediction of the splicing pattern of any primary transcript from its sequence. Outside of the core splice site motifs, the bulk of the information required for splicing is thought to be contained in exonic and intronic cis-regulatory elements that function by recruitment of sequence-specific RNA-binding protein factors that either activate or repress the use of adjacent splice sites. Here, we summarize the current state of knowledge of splicing cis-regulatory elements and their context-dependent effects on splicing, emphasizing recent global/genome-wide studies and open questions.Keywords: context dependence; pre-mRNA splicing; splicing code; splicing factor; splicing regulation PRE-MRNA SPLICING Because human genes typically contain multiple introns, the process of pre-mRNA splicing is an essential step in the expression of most genes. A majority of human genes undergo alternative splicing (AS), generating multiple splicing isoforms containing different combinations of exons (Johnson et al. 2003). The effects of AS on protein products can be dramatic, e.g., producing soluble versus membranebound forms of the Fas receptor that have opposing effects on apoptosis (Cascino et al. 1995), or producing isoforms of the Drosophila fruitless protein that act to specify sexual orientation (Demir and Dickson 2005). The major forms of AS are summarized in Figure 1A. Splicing regulation has been comprehensively described in several recent reviews (Black 2003;Konarska and Query 2005;Matlin et al. 2005;Blencowe 2006;House and Lynch 2008). Here, we briefly summarize some aspects of splicing specificity and regulation before turning to our main topic of splicing regulatory elements and the rules governing their activity.The sequential phosphodiester transfer reactions involved in splicing are catalyzed by large ribonucleoprotein complexes known as spliceosomes. Containing more than 100 core proteins and five small nuclear RNAs (snRNAs U1, U2, U4, U5, and U6), spliceosomes may be the most complex machines in the cell (Zhou et al. 2002;Jurica and Moore 2003;Nilsen 2003). In addition to these core factors, additional regulatory proteins participate in the splicing of particular pre-mRNAs. Splicing of most introns is thought to occur cotranscriptionally with fairly extensive interactions between splicing factors and the core transcription machinery ( CORE SPLICING SIGNALSThree sites, the 59 splice site (59ss), the 39 splice site (39ss), and the branch point sequence (BPS), participate in the splicing reaction and are present in every intron, and thus are known as the core splicing signals. These signals are recognized multiple times during spl...

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“…This enhancement can occur through recruitment of the spliceosomal machinery or by antagonism of a splicing repressor protein (Long and Caceres 2009;Shepard and Hertel 2009). SR proteins can also repress alternative splicing through multiple mechanisms, including acting through intronic splicing silencer elements (ISSs) adjacent to an alternative exon (Kanopka et al 1996;Buratti et al 2007). Modulation of SR proteins by small molecules has been shown in some studies, and they present interesting therapeutic targets for misregulated alternative splicing events (Soret et al 2005;Katzenberger et al 2009).…”

Section: Introductionmentioning

confidence: 99%

The cardiotonic steroid digitoxin regulates alternative splicing through depletion of the splicing factors SRSF3 and TRA2B

Anderson

Lin²,

Xiao³

et al. 2012

RNA

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Modulation of alternative pre-mRNA splicing is a potential approach to therapeutic targeting for a variety of human diseases. We investigated the mechanism by which digitoxin, a member of the cardiotonic steroid class of drugs, regulates alternative splicing. Transcriptome-wide analysis identified a large set of alternative splicing events that change after digitoxin treatment. Within and adjacent to these regulated exons, we identified enrichment of potential binding sites for the splicing factors SRp20 (SRSF3/SFRS3) and Tra2-b (SFRS10/TRA2B). We further find that both of these proteins are depleted from cells by digitoxin treatment. Characterization of SRp20 and Tra2-b splicing targets revealed that many, but not all, digitoxin-induced splicing changes can be attributed to the depletion of one or both of these factors. Re-expression of SRp20 or Tra2-b after digitoxin treatment restores normal splicing of their targets, indicating that the digitoxin effect is directly due to these factors. These results demonstrate that cardiotonic steroids, long prescribed in the clinical treatment of heart failure, have broad effects on the cellular transcriptome through these and likely other RNA binding proteins. The approach described here can be used to identify targets of other potential therapeutics that act as alternative splicing modulators.

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SR protein-mediated inhibition of CFTR exon 9 inclusion: molecular characterization of the intronic splicing silencer

Cited by 47 publications

References 75 publications

Mice with endogenous TDP ‐43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis

Mice with endogenous TDP ‐43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis

Splicing regulation: From a parts list of regulatory elements to an integrated splicing code

The cardiotonic steroid digitoxin regulates alternative splicing through depletion of the splicing factors SRSF3 and TRA2B

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