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Divergent levels of U1 snRNP proteins and U1 snRNA during phenotypic modulation. (A) Western blots for different splicing factors and RNA binding proteins in mouse aorta and bladder samples, comparing differentiated (D) and proliferative (P) samples. Acta2 and Myh-11 antibodies are markers of smooth muscle differentiation. Rpb1, Tubulin and Gapdh are loading controls. snRNA expression levels in mouse (B) and rat PAC1 cells (C) measured by qPCR. Primers for snRNA levels were normalized against the same set of genes as in Figures 5 and 6. Error bars represent standard deviation of the mean (n = 3). Statistically significance was calculated using Student's t-test, and is shown *P < 0.05. (D) RT-PCR for Snrp70 (qPCR with primers as in Figure 6) and Actn1 and Tpm1 (with primers as in Figure 2) in P and D Rat PAC1 cells. Error bars represent mean and standard deviation, (n = 3). (E) Left panels: RNA-FISH for U1 snRNA in rat PAC1 D and P cells. Right panels show DAPI staining for nuclei. (F) Immunofluorescence in rat PAC1 cells for SNRNP70 and U1C. Sm Actin is a marker of SMC differentiation.
Source publication
Alternative splicing (AS) is a key component of gene expression programs that drive cellular differentiation. Smooth muscle
cells (SMCs) are important in the function of a number of physiological systems; however, investigation of SMC AS has been
restricted to a handful of events. We profiled transcriptome changes in mouse de-differentiating SMCs a...
Contexts in source publication
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... Srsf1, a second IR event ( Figure 5C) showed atypical increased retention in proliferative cells. However, this intron lies within the 3 UTR and its splicing leads to NMD (54) (Supplementary Figure S7), so this event would also lead to downregulation of protein expression in the contractile phenotype cells. Notably, the behavior of this well documented AS-NMD event, also argues against global downregulation of NMD in differentiated cells. ...
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... we were able to detect some of these events in rat PAC1 VSMCs (31). We therefore tested whether cy- cloheximide treatment affected the ratio of spliced products in a manner consistent with inhibition of NMD (Supple- mentary Figure S7A) (52), and whether any of these RNA species were preferentially retained in the nucleus (Supple- mentary Figure S7B). We tested the effects of cyclohex- imide upon the non-productive CE events and also the 3 UTR IR event in Srsf1 (Supplementary Figure S7A). ...
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... we were able to detect some of these events in rat PAC1 VSMCs (31). We therefore tested whether cy- cloheximide treatment affected the ratio of spliced products in a manner consistent with inhibition of NMD (Supple- mentary Figure S7A) (52), and whether any of these RNA species were preferentially retained in the nucleus (Supple- mentary Figure S7B). We tested the effects of cyclohex- imide upon the non-productive CE events and also the 3 UTR IR event in Srsf1 (Supplementary Figure S7A). ...
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... therefore tested whether cy- cloheximide treatment affected the ratio of spliced products in a manner consistent with inhibition of NMD (Supple- mentary Figure S7A) (52), and whether any of these RNA species were preferentially retained in the nucleus (Supple- mentary Figure S7B). We tested the effects of cyclohex- imide upon the non-productive CE events and also the 3 UTR IR event in Srsf1 (Supplementary Figure S7A). Poi- son CE events in Sf3b3, Srsf7, Sfrs10 (Tra2 ) and Snrnp70 all showed apparent increased exon inclusion in response to cycloheximide. ...
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... results are consistent with the non- productive splicing patterns of these ASEs being linked to NMD. Interestingly, we observed cycloheximide stabi- lization of a non-productive Snrnpa1 product with exon 6 skipped in PAC1 cells (Supplementary Figure S7A). In mouse primary samples we had observed retention of both introns flanking exon 6, but not the exon 6 skipping event. ...
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... but one of the detected IR events were preferentially nuclear retained (Supplementary Figure S7B). The only ex- ception was Srsf1 intron 5, the sole IR event that was up- regulated in proliferative SMCs ( Figure 5C) and an intron whose splicing leads to NMD. ...
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... both the non-productive Snrnp70 splicing events showed some nu- clear retention. The poison CE was partially retained in the nucleus, while the annotated alternative polyA event was highly nuclear retained, suggesting that the detected RNA probably corresponds to IR (Supplementary Figure S7B). Of note, inclusion of the Snrnp70 poison CE results in an RNA that is both preferentially nuclear retained as well as an NMD substrate. ...
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... retained intron in CLK1 can be post- transcriptionally spliced in response to heat or osmotic stress or upon administration of small molecule CLK inhibitors (57,59). Consistent with this we observed that addition of the CLK inhibitor TG003 (20 M) (59) to PAC1 cells led to rapid splicing of the retained CLK1 in- tron, but none of the other IR events that we had detected (Supplementary Figure S7C), indicating that the SMC IR events are not all under a common mode of control with CLK1. ...
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... splicing of RNA binding proteins frequently occurs in re- sponse to high levels of the cognate protein as a result of negative feedback loops (52,60-63)). To address whether this is the case in the differentiated SMCs, we carried out western blots on protein extracted from differentiated and proliferative SMCs from mouse aorta and bladder ( Figure 7A). While the levels of markers for contractile SMCs were higher in the differentiated compared to proliferative cells (Myh11, Acta2), the levels of the various splicing factors and RNA binding proteins were either higher in prolifer- ative cells (Snrnp70, Snrnpa1, Sf3b1, Srsf6, Srsf7, Rbm3) or were not altered substantially. ...
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... view of the variation in levels of core splicing fac- tors, including snRNP proteins, we analyzed the levels of the spliceosomal snRNAs ( Figure 7B). Remarkably, qRT- PCR analysis showed that levels of U1, U2, U4 and U5 snRNAs were all substantially higher in differentiated than proliferative aorta SMCs. ...
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... largest difference was ob- served for U1 snRNA, with 4.5-fold higher levels in dif- ferentiated cells. The elevated U1 snRNA levels contrasts with the lower levels of the U1 snRNP proteins U1 70K (Snrnp70) and U1C in differentiated aorta samples ( Figure 7A). To pursue this observation further, we used cultured rat PAC1 SMCs which had been serially passaged under conditions to promote more differentiated or more prolif- erative phenotypes (Figure 7C-E). ...
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... elevated U1 snRNA levels contrasts with the lower levels of the U1 snRNP proteins U1 70K (Snrnp70) and U1C in differentiated aorta samples ( Figure 7A). To pursue this observation further, we used cultured rat PAC1 SMCs which had been serially passaged under conditions to promote more differentiated or more prolif- erative phenotypes (Figure 7C-E). RT-qPCR showed 4-5 fold higher levels of U1 snRNA and 2.5-fold higher levels of U4 snRNA in differentiated compared to more prolifer- ative PAC1 cells, while levels of U2, U5 and U6 did not dif- fer significantly ( Figure 7C). ...
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... pursue this observation further, we used cultured rat PAC1 SMCs which had been serially passaged under conditions to promote more differentiated or more prolif- erative phenotypes (Figure 7C-E). RT-qPCR showed 4-5 fold higher levels of U1 snRNA and 2.5-fold higher levels of U4 snRNA in differentiated compared to more prolifer- ative PAC1 cells, while levels of U2, U5 and U6 did not dif- fer significantly ( Figure 7C). Analysis of ASEs in Snrnp70, Actn1 and Tpm1, showed changes as had been observed in the mouse primary cells; in particular Snrnp70 showed higher levels of non-productive splicing in the differenti- ated cells ( Figure 7D). ...
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... showed 4-5 fold higher levels of U1 snRNA and 2.5-fold higher levels of U4 snRNA in differentiated compared to more prolifer- ative PAC1 cells, while levels of U2, U5 and U6 did not dif- fer significantly ( Figure 7C). Analysis of ASEs in Snrnp70, Actn1 and Tpm1, showed changes as had been observed in the mouse primary cells; in particular Snrnp70 showed higher levels of non-productive splicing in the differenti- ated cells ( Figure 7D). RNA-FISH images also consistently showed higher levels of U1 snRNA in differentiated com- pared to proliferative PAC1 cells ( Figure 7E). ...
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... of ASEs in Snrnp70, Actn1 and Tpm1, showed changes as had been observed in the mouse primary cells; in particular Snrnp70 showed higher levels of non-productive splicing in the differenti- ated cells ( Figure 7D). RNA-FISH images also consistently showed higher levels of U1 snRNA in differentiated com- pared to proliferative PAC1 cells ( Figure 7E). In contrast, immunofluorescence microscopy showed levels of U1 70K (Snrnp70) to be lower in differentiated compared to prolif- erative PAC1 cells (Figure 7F), paralleling the changes in non-productive splicing ( Figure 7C). ...
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... images also consistently showed higher levels of U1 snRNA in differentiated com- pared to proliferative PAC1 cells ( Figure 7E). In contrast, immunofluorescence microscopy showed levels of U1 70K (Snrnp70) to be lower in differentiated compared to prolif- erative PAC1 cells (Figure 7F), paralleling the changes in non-productive splicing ( Figure 7C). These alterations in stoichiometry of U1 snRNP components could have impor- tant consequences for the various functions of U1 snRNP, including in regulation of AS. ...
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... images also consistently showed higher levels of U1 snRNA in differentiated com- pared to proliferative PAC1 cells ( Figure 7E). In contrast, immunofluorescence microscopy showed levels of U1 70K (Snrnp70) to be lower in differentiated compared to prolif- erative PAC1 cells (Figure 7F), paralleling the changes in non-productive splicing ( Figure 7C). These alterations in stoichiometry of U1 snRNP components could have impor- tant consequences for the various functions of U1 snRNP, including in regulation of AS. ...
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... suggests that PTB com- monly acts to repress SMC-specific exons outside of differ- entiated cells, as has been shown for the SM exon of Actn1 (14,15). Consistent with this, western blots indicated that PTBP1 levels were lower in aorta and bladder differentiated cells than in cultured cells (Figure 7). Nevertheless, knock- down of PTBP1/2 in PAC1 cells was not sufficient to switch splicing patterns fully to the levels of exon inclusion seen in mouse tissues (Figures 2 and 4), suggesting that other mechanisms might be involved in countering repression by PTBP1. ...
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... were unable to address which pathways were used in the differentiated mouse tissue samples used for the array analysis. However, using PAC1 cells we showed that some of the poison CE events were re- sponsive to cycloheximide, consistent with degradation by the translation-dependent NMD pathway, while most of the IR events were preferentially nuclear retained (Supplemen- tary Figure S7). Strikingly, the one case of IR that increased in proliferative cells was a 3 UTR intron in Srsf1 ( Figure 5C). ...
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... non-productive splicing events within the genes for splicing factors and other RBPs are able to operate as neg- ative feedback loops that act to prevent over-expression of the cognate factor, often via NMD (52,60-63). This is not the case in SMCs, where we observed lower levels of the corresponding protein accompanying the non-productive splicing in differentiated cells (Figure 7). This suggests that in this case the non-productive splicing responds to signal- ing that actively downregulates the steady state level of pro- teins, possibly re-setting the level maintained by negative feedback (73). ...
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... physiologi- cal alteration in the levels of the U2 snRNP protein Sf3b1 affects specific ASEs with clear functional consequences (82). We observed reductions in the levels of U1 (U1 70K) and U2 snRNP (Snrpa1, Sf3b1) proteins in aorta and blad- der differentiated cells (Figure 7A), all of which were seen to affect multiple ASEs upon knockdown (80). Strikingly, the levels of the cognate snRNAs did not change in paral- lel. ...
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... the levels of the cognate snRNAs did not change in paral- lel. This was most obvious for U1 snRNP where the levels of U1 snRNA and U1 snRNP proteins changed in opposite di- rections between differentiated and proliferative cells (Fig- ure 7B-D). We cannot currently say whether the differenti- ated cells have an excess of U1 snRNA over U1 70K protein (encoded by Snrnp70), or whether proliferative cells have an excess of U1 70K over U1 snRNA. ...
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Citations
... Among all AS events, IR received a minimal attention and considered to be a rare event in which hydrolysis often occurs via the cellular degradation pathway. However, growing number of reports suggest that IR could affect 80% of the coding genes (Middleton et al., 2017), especially those involved in cell differentiation (Llorian et al., 2016) and cell cycling (Braunschweig et al., 2014;Llorian et al., 2016;Middleton et al., 2017). Per the cancer genomic atlas (TSGA) and the transcriptomic cancer studies, IR is considered the common AS mode among all cancer types as it accounts for the wide diversities in cancer transcriptomes (Supek et al., 2014;Dvinge and Bradley, 2015). ...
... Among all AS events, IR received a minimal attention and considered to be a rare event in which hydrolysis often occurs via the cellular degradation pathway. However, growing number of reports suggest that IR could affect 80% of the coding genes (Middleton et al., 2017), especially those involved in cell differentiation (Llorian et al., 2016) and cell cycling (Braunschweig et al., 2014;Llorian et al., 2016;Middleton et al., 2017). Per the cancer genomic atlas (TSGA) and the transcriptomic cancer studies, IR is considered the common AS mode among all cancer types as it accounts for the wide diversities in cancer transcriptomes (Supek et al., 2014;Dvinge and Bradley, 2015). ...
Human transcriptome can undergo RNA mis-splicing due to spliceopathies contributing to the increasing number of genetic diseases including muscular dystrophy (MD), Alzheimer disease (AD), Huntington disease (HD), myelodysplastic syndromes (MDS). Intron retention (IR) is a major inducer of spliceopathies where two or more introns remain in the final mature mRNA and account for many intronic expansion diseases. Potential removal of such introns for therapeutic purposes can be feasible when utilizing bioinformatics, catalytic RNAs, and nano-drug delivery systems. Overcoming delivery challenges of catalytic RNAs was discussed in this review as a future perspective highlighting the significance of utilizing synthetic biology in addition to high throughput deep sequencing and computational approaches for the treatment of mis-spliced transcripts.
... For instance, transformer 2β (Tra2β) ensures the splicing of MYPT1, a gene involved in SMC differentiation from slow to fast contractile phenotype, allowing the vasorelaxation in blood vessels [83,84]. Polypyrimidine Tract Binding protein 1 (PTBP1) mediates alternative splicing in SMCs, resulting in a contractile phenotype [85]. DEAD-Box-Protein 5 (DDX-5) directly binds and maintains GATA-6 expression, a pivotal factor for maintaining SMCs contractile phenotype [86]. ...
Atherosclerotic cardiovascular disease (ASCVD) remains the major cause of premature death and disability worldwide, even when patients with an established manifestation of atherosclerotic heart disease are optimally treated according to the clinical guidelines. Apart from the epigenetic control of transcription of the genetic information to messenger RNAs (mRNAs), gene expression is tightly controlled at the post-transcriptional level before the initiation of translation. Although mRNAs are traditionally perceived as the messenger molecules that bring genetic information from the nuclear DNA to the cytoplasmic ribosomes for protein synthesis, emerging evidence suggests that processes controlling RNA metabolism, driven by RNA-binding proteins (RBPs), affect cellular function in health and disease. Over the recent years, vascular endothelial cell, smooth muscle cell and immune cell RBPs have emerged as key co- or post-transcriptional regulators of several genes related to vascular inflammation and atherosclerosis. In this review, we provide an overview of cell-specific function of RNA-binding proteins involved in all stages of ASCVD and how this knowledge may be used for the development of novel precision medicine therapeutics.
... In smooth muscle, studies have begun to associate alternative splicing changes with smooth muscle's ability to (1) remain phenotypically plastic, allowing for dedifferentiation from a contractile phenotype to a proliferative one, and (2) vary in cell type depending on functional need, such as producing tonic (slow) contractile cells that maintain contractions over time or phasic (fast) contractile cells that undergo strong contractions followed by relaxation (Figure 4a) [126,127]. Bioinformatic assessment of transcript splicing from mouse exon junction arrays identified 4244 alternative splicing events between proliferative and differentiated aortic (tonic) smooth muscle and 5193 alternative splicing events between proliferative and differentiated bladder (phasic) smooth muscle, suggesting that alternative splicing plays a key role in determining and/or maintaining SMC state and type [128]. Alternative splicing of mRNA encoding contractile network proteins, such as alpha-tropomyosin (Tpm1), alpha-actinin-1 (Actn1), myocardin (Myocd), and vinculin (Vcl), have been shown to switch during differentiation, thus altering SMC differentiation state and contractile ability [128][129][130][131]. ...
... Bioinformatic assessment of transcript splicing from mouse exon junction arrays identified 4244 alternative splicing events between proliferative and differentiated aortic (tonic) smooth muscle and 5193 alternative splicing events between proliferative and differentiated bladder (phasic) smooth muscle, suggesting that alternative splicing plays a key role in determining and/or maintaining SMC state and type [128]. Alternative splicing of mRNA encoding contractile network proteins, such as alpha-tropomyosin (Tpm1), alpha-actinin-1 (Actn1), myocardin (Myocd), and vinculin (Vcl), have been shown to switch during differentiation, thus altering SMC differentiation state and contractile ability [128][129][130][131]. These specific splicing events occur during SMC differentiation across SMC cell types. ...
Myotonic dystrophy (DM) is a highly variable, multisystemic disorder that clinically affects one in 8000 individuals. While research has predominantly focused on the symptoms and pathological mechanisms affecting striated muscle and brain, DM patient surveys have identified a high prevalence for gastrointestinal (GI) symptoms amongst affected individuals. Clinical studies have identified chronic and progressive dysfunction of the esophagus, stomach, liver and gallbladder, small and large intestine, and rectum and anal sphincters. Despite the high incidence of GI dysmotility in DM, little is known regarding the pathological mechanisms leading to GI dysfunction. In this review, we summarize results from clinical and molecular analyses of GI dysfunction in both genetic forms of DM, DM type 1 (DM1) and DM type 2 (DM2). Based on current knowledge of DM primary pathological mechanisms in other affected tissues and GI tissue studies, we suggest that misregulation of alternative splicing in smooth muscle resulting from the dysregulation of RNA binding proteins muscleblind-like and CUGBP-elav-like is likely to contribute to GI dysfunction in DM. We propose that a combinatorial approach using clinical and molecular analysis of DM GI tissues and model organisms that recapitulate DM GI manifestations will provide important insight into defects impacting DM GI motility.
... Tissue-specific expression of MP regulatory subunits imparts properties onto MP that are specific to smooth muscle contractile subtypes and distinct from MP in nonmuscle cells, in which it mediates shape changes and motility (4,5). A great deal of phenotypic diversity in SMCs is generated by alternative exon usage (6)(7)(8), a process that is nearly universal in mRNA precursors (9). For only a few of the thousands of regulated alternative splicing events in SMCs is the functional significance understood. ...
... We and others have demonstrated that the splicing of Mypt1 E24 is tissue-specific: Mypt1 E24 is predominantly included in phasic smooth muscle such as bladder with more exon skipping in tonic smooth muscle such as aorta, and completely skipped in proliferative smooth muscle and nonmuscle cells (reviewed in Refs. 3,18; see also Ref. 7). The splicing of Mypt1 E24 is also developmentally regulated and modulates in disease. ...
Alternative splicing of exon24 (E24) of Myosin phosphatase targeting subunit 1 (Mypt1) by setting sensitivity to NO/cGMP mediated relaxation is a key determinant of smooth muscle function. Here we defined expression of myosin phosphatase (MP) subunits and isoforms by creation of new genetic mouse models, assay of human and mouse tissues and query of public databases. A Mypt1-LacZ reporter mouse revealed that Mypt1 transcription is turned on early in development during smooth muscle differentiation. Mypt1 is not as tightly restricted in its expression as smooth muscle myosin heavy chain (Myh11) and its E6 splice variant. Mypt1 is enriched in mature smooth vs non-muscle cells. The E24 splice variant and leucine zipper minus protein isoform which it encodes is enriched in phasic vs tonic smooth muscle. In the vascular system, E24 splicing increases as vessel size decreases. In the gastrointestinal system E24 splicing is most predominant in smooth muscle of the small intestine. Tissue-specific expression of MP subunits and Mypt1 E24 splicing is conserved in humans, while a splice variant of the inhibitory subunit (CPI-17) is unique to humans. A Mypt1 E24 mini-gene splicing reporter mouse generated to define patterns of E24 splicing in SMCs dispersed throughout the organ systems was unsuccessful. In summary, expression of Mypt1 and splicing of E24 is part of the program of smooth muscle differentiation, is further enhanced in phasic smooth muscle, and is conserved in humans. Its low-level expression in non-muscle cells may confound its measurement in tissue samples.
... A number of studies on transcriptome-wide analyses have demonstrated that different IR events control gene expression programs during both normal development and disease-related states. For example, increased IR incidences were observed in various cells of the hematopoietic lineage, such as transformation of erythroblasts, megakaryocytes, granulocytes and CD4 T-cells (Wong et al. 2013;Pimentel et al. 2016;Edwards et al. 2016;Ni et al. 2016), during differentiation of ESCs into neural progenitors, reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells (IPSCs) (Braunschweig et al. 2014;Hussein et al. 2014;Boutz et al. 2015), and during normal development of smooth muscle cells (Llorian et al. 2016). Comparative analysis of these studies has revealed the common and unique features of IR-mediated regulation including its tissue-and cell-type specificities. ...
... However, a high percentage of IR events was not necessary for the induction of terminal differentiation. More plastic cell types whose functional maturation involves transition to proliferative states exhibit decreased IR incidence as shown during dedifferentiation of smooth muscle cells (Wong et al. 2013;Cho et al. 2014;Braunschweig et al. 2014;Pimentel et al. 2016;Edwards et al. 2016;Llorian et al. 2016). Some differentiation programs involve more complex patterns of IR for example, the final stages of erythropoiesis include a progressive increase in IR events before achieving a terminal stage that exhibited lower IR than the precursor cells. ...
... Elimination of these transcripts was achieved through cytoplasmic NMD (Wong et al. 2013). Such a function of IR in transcriptome tuning remains in agreement with a number of other reports, including maturation of cells of the hematopoietic lineage (Yap et al. 2012;Boutz et al. 2015;Pimentel et al. 2016;Edwards et al. 2016;Llorian et al. 2016;Mauger et al. 2016). ...
The discovery of introns over four decades ago revealed a new vision of genes and their interrupted arrangement. Throughout the years, it has appeared that introns play essential roles in the regulation of gene expression. Unique processing of excised introns through the formation of lariats suggests a widespread role for these molecules in the structure and function of cells. In addition to rapid destruction, these lariats may linger on in the nucleus or may even be exported to the cytoplasm, where they remain stable circular RNAs (circRNAs). Alternative splicing (AS) is a source of diversity in mature transcripts harboring retained introns (RI-mRNAs). Such RNAs may contain one or more entire retained intron(s) (RIs), but they may also have intron fragments resulting from sequential excision of smaller subfragments via recursive splicing (RS), which is characteristic of long introns. There are many potential fates of RI-mRNAs, including their downregulation via nuclear and cytoplasmic surveillance systems and the generation of new protein isoforms with potentially different functions. Various reports have linked the presence of such unprocessed transcripts in mammals to important roles in normal development and in disease-related conditions. In certain human neurological-neuromuscular disorders, including myotonic dystrophy type 2 (DM2), frontotemporal dementia/amyotrophic lateral sclerosis (FTD/ALS) and Duchenne muscular dystrophy (DMD), peculiar processing of long introns has been identified and is associated with their pathogenic effects. In this review, we discuss different mechanisms involved in the processing of introns during AS and the functions of these large sections of the genome in our biology.
... Coinciding with significant technological and conceptual innovations in RNA biology, there has been a growing focus on co-transcriptional processes that regulate gene expression in VSMCs. In particular, alternative splicing (AS) of mRNAs and its regulatory factors have been investigated in a variety of physiological and pathological VSMC contexts [18][19][20][21][22][23][24][25]. Collectively, these studies have revealed an expanding role of AS and splicing factors in promoting protein diversity and regulating gene expression in VSMC biology. ...
... Although initially dismissed as an aberration, IR is now established as a key mechanism of gene expression control in many cell types, particularly in the neuronal and haematopoietic lineages and more recently in VSMCs [21,[41][42][43][44][45][46][47][48]. IR can result in posttranscriptional gene repression, as many introns contains premature termination codons which facilitate cytoplasmic nonsense-mediated decay [47,49]. ...
... These regulatory proteins, along with other RNA binding proteins (RBPs), play essential roles in either enhancing or repressing spliceosome formation at specific sites on nascent mRNA [38]. In the context of AS in VSMC biology, several studies have recently explored the ways specific RBPs and splicing factors influence VSMC function in health and disease [18][19][20][21][22][23][24][25]. Although initially dismissed as an aberration, IR is now established as a key mechanism of gene expression control in many cell types, particularly in the neuronal and haematopoietic lineages and more recently in VSMCs [21,[41][42][43][44][45][46][47][48]. ...
Vascular smooth muscle cells (VSMCs) display extraordinary phenotypic plasticity. This allows them to differentiate or dedifferentiate, depending on environmental cues. The ability to ‘switch’ between a quiescent contractile phenotype to a highly proliferative synthetic state renders VSMCs as primary mediators of vascular repair and remodelling. When their plasticity is pathological, it can lead to cardiovascular diseases such as atherosclerosis and restenosis. Coinciding with significant technological and conceptual innovations in RNA biology, there has been a growing focus on the role of alternative splicing in VSMC gene expression regulation. Herein, we review how alternative splicing and its regulatory factors are involved in generating protein diversity and altering gene expression levels in VSMC plasticity. Moreover, we explore how recent advancements in the development of splicing-modulating therapies may be applied to VSMC-related pathologies.
... The resulting transcripts may be subject to frame shifts, premature termination codons and/or non-sense mediated decay (NMD) (18,19). Collectively, >90% of human multi-exon genes are subject to alternative splicing which greatly expands the diversity and function of the proteome (20,21). In eukaryotes the spliceosome, where socalled splice factors (SFs) cooperate with five small nuclear ribonucleoprotein complexes (U1, U2, U4/U6, and U5), recognizes and assembles on introns to cleave and ligate RNA molecules for intron removal, generating protein-coding mRNAs (22). ...
Gain-of-function mutations of the TLR adaptor and oncoprotein MyD88 drive B cell lymphomagenesis via sustained NF-κB activation. In myeloid cells, both short and sustained TLR activation and NF-κB activation lead to the induction of inhibitory MYD88 splice variants that restrain prolonged NF-κB activation. We therefore sought to investigate whether such a negative feedback loop exists in B cells. Analyzing MYD88 splice variants in normal B cells and different primary B cell malignancies, we observed that MYD88 splice variants in transformed B cells are dominated by the canonical, strongly NF-κB-activating isoform of MYD88 and contain at least three novel, so far uncharacterized signaling-competent splice isoforms. Sustained TLR stimulation in B cells unexpectedly reinforces splicing of NF-κB-promoting, canonical isoforms rather than the ‘MyD88s’, a negative regulatory isoform reported to be typically induced by TLRs in myeloid cells. This suggests that an essential negative feedback loop restricting TLR signaling in myeloid cells at the level of alternative splicing, is missing in B cells when they undergo proliferation, rendering B cells vulnerable to sustained NF-κB activation and eventual lymphomagenesis. Our results uncover MYD88 alternative splicing as an unappreciated promoter of B cell lymphomagenesis and provide a rationale why oncogenic MYD88 mutations are exclusively found in B cells.
... This statement is also supported by the Green and colleague's research as the genes that encoded the regulators of macrophage transcription, signaling inflammation, and phagocytosis has increased their expression when the IR events decreased [55]. As it is known, that intron retention (IR) is the process where instead of typically being spliced out, the introns remain intact in the mature mRNAs and thus whole process of IR supposedly has numerous physiological drawbacks resulting in different diseases [47,48].Currently, many researchers strongly claim that IR is a key mechanism to control gene expression during the development, differentiation and activation of several types of mammalian cell [56][57][58][59][60][61][62][63]. A recent study by Green and colleague claimed that intron retention affected the expression of key genes (ID2, IRF7, ENG, and L AT) involved in the development and function of macrophages those are the key inflammatory regulator [55]. ...
Cancer and inflammation are connected by intrinsic pathways and extrinsic pathway where the intrinsic pathway is activated by genetic events including mutation, chromosomal rearrangement or amplification, and the inactivation of tumor-suppressor genes, as well as the extrinsic pathway, is the inflammatory or infectious conditions that increase the cancer risk. On the other hand, introns are non-coding elements of the genome and play a functional role to generate more gene products through splicing out, transcription, polyadenylation, mRNA export, and translation. Moreover, introns also may act as a primary element of some of the most highly expressed genes in the genome. Intron may contain their regulatory function as CRISPR system which is activated after the demand of specific gene for specific protein formation where those are required for gene expression, they go for transcription and rest of them form splicing. This chapter will focus on the plausible role of introns to influence the genetic events of inflammation-mediated cancer cell development.
... The specificity of the antibodies used in this study had either been tested and reported previously (Yang et al., 2014;Llorian et al., 2016;Wang et al., 2016) or was examined using Western blotting ( Figure 1I; using a method published previously; Palpagama et al., 2019a,b;Pandya et al., 2019). Mice were euthanized by cervical dislocation and the brains rapidly removed. ...
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were accompanied by cognitive deterioration, as demonstrated by long-term spatial
memory impairment. This study is reporting a comprehensive examination of a complex
set of parameters associated with intrahippocampal administration of Aβ1–42 in mice and their spatiotemporal interactions and combined contribution to the disease progression. We show that a single Aβ injection can reproduce aspects of the inflammatory, vascular,
and p-tau induced pathology occurring in the AD human brain that lead to cognitive
deficits.
... The expression level of PTBP1 is associated with neuronal development and muscle differentiation [56,133]. Wide analyses of PTB targets in HeLa cells indicate that PTBP1 represses many neuronal and striated muscle-specific exons in genes encoding cytoskeletal and signaling proteins, highlighting its role in the fine regulation of protein isoform expression required for cell differentiation [134][135][136]. ...
Alternative splicing is a regulatory mechanism essential for cell differentiation and tissue organization. More than 90% of human genes are regulated by alternative splicing events, which participate in cell fate determination. The general mechanisms of splicing events are well known, whereas only recently have deep-sequencing, high throughput analyses and animal models provided novel information on the network of functionally coordinated, tissue-specific, alternatively spliced exons. Heart development and cardiac tissue differentiation require thoroughly regulated splicing events. The ribonucleoprotein RBM20 is a key regulator of the alternative splicing events required for functional and structural heart properties, such as the expression of TTN isoforms. Recently, the polypyrimidine tract-binding protein PTBP1 has been demonstrated to participate with RBM20 in regulating splicing events. In this review, we summarize the updated knowledge relative to RBM20 and PTBP1 structure and molecular function; their role in alternative splicing mechanisms involved in the heart development and function; RBM20 mutations associated with idiopathic dilated cardiovascular disease (DCM); and the consequences of RBM20-altered expression or dysfunction. Furthermore, we discuss the possible application of targeting RBM20 in new approaches in heart therapies.
