scholarly journals U2AF65-Dependent SF3B1 Function in SMN Alternative Splicing

Cells ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 2647
Author(s):  
Namjeong Choi ◽  
Yongchao Liu ◽  
Jagyeong Oh ◽  
Jiyeon Ha ◽  
Xuexiu Zheng ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Site Selection ◽  
Muscular Atrophy ◽  
Survival Motor Neuron ◽  
Pcr Analysis ◽  
Splice Site Selection ◽  
Interaction Domain ◽  
Regulatory Functions ◽  
Auxiliary Factor

Splicing factor 3b subunit 1 (SF3B1) is an essential protein in spliceosomes and mutated frequently in many cancers. While roles of SF3B1 in single intron splicing and roles of its cancer-linked mutant in aberrant splicing have been identified to some extent, regulatory functions of wild-type SF3B1 in alternative splicing (AS) are not well-understood yet. Here, we applied RNA sequencing (RNA-seq) to analyze genome-wide AS in SF3B1 knockdown (KD) cells and to identify a large number of skipped exons (SEs), with a considerable number of alternative 5′ splice-site selection, alternative 3′ splice-site selection, mutually exclusive exons (MXE), and retention of introns (RI). Among altered SEs by SF3B1 KD, survival motor neuron 2 (SMN2) pre-mRNA exon 7 splicing was a regulatory target of SF3B1. RT-PCR analysis of SMN exon 7 splicing in SF3B1 KD or overexpressed HCT116, SH-SY5Y, HEK293T, and spinal muscular atrophy (SMA) patient cells validated the results. A deletion mutation demonstrated that the U2 snRNP auxiliary factor 65 kDa (U2AF65) interaction domain of SF3B1 was required for its function in SMN exon 7 splicing. In addition, mutations to lower the score of the polypyrimidine tract (PPT) of exon 7, resulting in lower affinity for U2AF65, were not able to support SF3B1 function, suggesting the importance of U2AF65 in SF3B1 function. Furthermore, the PPT of exon 7 with higher affinity to U2AF65 than exon 8 showed significantly stronger interactions with SF3B1. Collectively, our results revealed SF3B1 function in SMN alternative splicing.

scholarly journals The Interaction and Colocalization of Sam68 with the Splicing-associated Factor YT521-B in Nuclear Dots Is Regulated by the Src Family Kinase p59fyn

1999 ◽  
Vol 10 (11) ◽  
pp. 3909-3926 ◽  
Author(s):  
Annette M. Hartmann ◽  
Oliver Nayler ◽  
Franz Werner Schwaiger ◽  
Axel Obermeier ◽  
Stefan Stamm
Keyword(s):  
Glutamic Acid ◽  
Splice Site ◽  
Site Selection ◽  
Dependent Manner ◽  
Src Family Kinase ◽  
Cell Type Specificity ◽  
Splice Site Selection ◽  
Interaction Domain ◽  
Concentration Dependent Manner ◽  
Amino Terminal

Alternative pre-mRNA splicing patterns can change an extracellular stimulus, but the signaling pathways leading to these changes are still poorly characterized. Here, we describe a tyrosine-phosphorylated nuclear protein, YT521-B, and show that it interacts with the nuclear transcriptosomal component scaffold attachment factor B, and the 68-kDa Src substrate associated during mitosis, Sam68. Northern blot analysis demonstrated ubiquitous expression, but detailed RNA in situ analysis revealed cell type specificity in the brain. YT521-B protein is localized in the nucleoplasm and concentrated in 5–20 large nuclear dots. Deletion analysis demonstrated that the formation of these dots depends on the presence of the amino-terminal glutamic acid-rich domain and the carboxyl-terminal glutamic acid/arginine-rich region. We show that the latter comprises an important protein–protein interaction domain. The Src family kinase p59fyn-mediated tyrosine phosphorylation of Sam68 negatively regulates its association with YT521-B, and overexpression of p59fyn dissolves nuclear dots containing YT521-B. In vivo splicing assays demonstrated that YT521-B modulates alternative splice site selection in a concentration-dependent manner. Together, our data indicate that YT521-B and Sam68 may be part of a signal transduction pathway that influences splice site selection.

scholarly journals Origin, Splicing, and Expression of Rodent Amelogenin Exon 8

2006 ◽  
Vol 85 (10) ◽  
pp. 894-899 ◽  
Author(s):  
J.D. Bartlett ◽  
R. L. Ball ◽  
T. Kawai ◽  
C.E. Tye ◽  
M. Tsuchiya ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Site Selection ◽  
Proteolytic Processing ◽  
Rt Pcr ◽  
Splice Site Selection ◽  
Rna Transcripts ◽  
Splicing Pattern ◽  
Alternatively Spliced ◽  
Gene Structures

Amelogenin RNA transcripts undergo extensive alternative splicing, and MMP-20 processes the isoforms following their secretion. Since amelogenins have been ascribed cell-signaling activities, we asked if a lack of proteolytic processing by MMP-20 affects amelogenin signaling and consequently alters amelogenin splice site selection. RT-PCR analyses of amelogenin mRNA between control and Mmp20− /−mice revealed no differences in the splicing pattern. We characterized 3 previously unidentified amelogenin alternatively spliced transcripts and demonstrated that exon-8-encoded amelogenin isoforms are processed by MMP-20. Transcripts with exon 8 were expressed approximately five-fold less than those with exon 7. Analyses of the mouse and rat amelogenin gene structures confirmed that exon 8 arose in a duplication of exons 4 through 5, with translocation of the copy downstream of exon 7. No downstream genomic sequences homologous to exons 4–5 were present in the bovine or human amelogenin genes, suggesting that this translocation occurred only in rodents.

scholarly journals mTOR-regulated U2af1 tandem exon splicing specifies transcriptome features for translational control

2019 ◽  
Vol 47 (19) ◽  
pp. 10373-10387 ◽  
Author(s):  
Jae-Woong Chang ◽  
Hsin-Sung Yeh ◽  
Meeyeon Park ◽  
Luke Erber ◽  
Jiao Sun ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Translational Control ◽  
Transcriptome Profiling ◽  
Molecular Profile ◽  
Splice Site Selection ◽  
Exon Splicing ◽  
Alternative Splicing Events ◽  
U2 Auxiliary Factor ◽  
Auxiliary Factor

Abstract U2 auxiliary factor 1 (U2AF1) functions in 3′-splice site selection during pre-mRNA processing. Alternative usage of duplicated tandem exons in U2AF1 produces two isoforms, U2AF1a and U2AF1b, but their functional differences are unappreciated due to their homology. Through integrative approaches of genome editing, customized-transcriptome profiling and crosslinking-mediated interactome analyses, we discovered that the expression of U2AF1 isoforms is controlled by mTOR and they exhibit a distinctive molecular profile for the splice site and protein interactomes. Mechanistic dissection of mutually exclusive alternative splicing events revealed that U2AF1 isoforms’ inherent differential preferences of nucleotide sequences and their stoichiometry determine the 3′-splice site. Importantly, U2AF1a-driven transcriptomes feature alternative splicing events in the 5′-untranslated region (5′-UTR) that are favorable for translation. These findings unveil distinct roles of duplicated tandem exon-derived U2AF1 isoforms in the regulation of the transcriptome and suggest U2AF1a-driven 5′-UTR alternative splicing as a molecular mechanism of mTOR-regulated translational control.

scholarly journals Role of the Modular Domains of SR Proteins in Subnuclear Localization and Alternative Splicing Specificity

1997 ◽  
Vol 138 (2) ◽  
pp. 225-238 ◽  
Author(s):  
Javier F. Cáceres ◽  
Tom Misteli ◽  
Gavin R. Screaton ◽  
David L. Spector ◽  
Adrian R. Krainer
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Site Selection ◽  
Cellular Localization ◽  
Sr Proteins ◽  
Dependent Manner ◽  
Splice Site Selection ◽  
Subnuclear Localization

SR proteins are required for constitutive pre-mRNA splicing and also regulate alternative splice site selection in a concentration-dependent manner. They have a modular structure that consists of one or two RNA-recognition motifs (RRMs) and a COOH-terminal arginine/serine-rich domain (RS domain). We have analyzed the role of the individual domains of these closely related proteins in cellular distribution, subnuclear localization, and regulation of alternative splicing in vivo. We observed striking differences in the localization signals present in several human SR proteins. In contrast to earlier studies of RS domains in the Drosophila suppressor-of-white-apricot (SWAP) and Transformer (Tra) alternative splicing factors, we found that the RS domain of SF2/ASF is neither necessary nor sufficient for targeting to the nuclear speckles. Although this RS domain is a nuclear localization signal, subnuclear targeting to the speckles requires at least two of the three constituent domains of SF2/ASF, which contain additive and redundant signals. In contrast, in two SR proteins that have a single RRM (SC35 and SRp20), the RS domain is both necessary and sufficient as a targeting signal to the speckles. We also show that RRM2 of SF2/ASF plays an important role in alternative splicing specificity: deletion of this domain results in a protein that, although active in alternative splicing, has altered specificity in 5′ splice site selection. These results demonstrate the modularity of SR proteins and the importance of individual domains for their cellular localization and alternative splicing function in vivo.

scholarly journals The second RNA-binding domain of the human splicing factor ASF/SF2 is the critical domain controlling adenovirus E1A alternative 5′-splice site selection

2004 ◽  
Vol 381 (2) ◽  
pp. 343-350 ◽  
Author(s):  
Vita DAUKSAITE ◽  
Göran AKUSJÄRVI
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Site Selection ◽  
Rna Binding ◽  
Splicing Factor ◽  
Rna Recognition Motif ◽  
Splice Site Selection ◽  
Critical Domain ◽  
Adenovirus E1a

The human splicing factor ASF/SF2 (alternative splicing factor/splicing factor 2) is modular in structure with two RNA-binding domains (RBD1 and RBD2) and a C-terminal domain rich in arginine–serine dipeptide repeats. ASF/SF2 is an essential splicing factor that also functions as an important regulator of alternative splicing. In adenovirus E1A (early region 1A) alternative pre-mRNA splicing, ASF/SF2 functions as a strong inducer of proximal 5′-splice-site selection, both in vitro and in vivo. In the present study, we tested the functional role of individual domains of ASF/SF2 in alternative splicing in vitro. We show that ASF/SF2-RBD2 is the critical domain controlling E1A alternative splicing. In fact, RBD2 alone is sufficient to mimic the activity of the full-length ASF/SF2 protein as an inducer of proximal 5′-splice-site selection in vitro. The RBD2 domain induces a switch to E1A-proximal 5′-splice-site usage by repressing distal 12 S splicing and simultaneously stimulates proximal 13 S splicing. In contrast, the ASF/SF2-RBD1 domain has a more general splicing enhancer phenotype and appears to stimulate preferentially cap-proximal 5′-splice-site selection. Furthermore, the SWQDLKD motif, which is conserved in all SR proteins (serine/arginine-rich proteins) containing two RBDs, and the ribonucleoprotein-1-type RNA recognition motif were both found to be necessary for the alternative splice-site-switching activity of ASF/SF2. The RNP-1 motif was necessary for efficient RNA binding, whereas the SWQDLKD motif most probably contributes by functioning as a surface-mediating critical protein–protein contact during spliceosome assembly.

SR protein kinases: the splice of life

1999 ◽  
Vol 77 (4) ◽  
pp. 293-298 ◽  
Author(s):  
David F Stojdl ◽  
John C Bell
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Site Selection ◽  
Developmental Process ◽  
Messenger Rnas ◽  
Splice Site Selection ◽  
Sr Protein ◽  
Specific Alternative ◽  
Mature Mrnas

The eukaryotic genome codes for most of its proteins though discontinuous coding sequences called exons, which are separated by noncoding sequences known as introns. Following transcription of a gene, these exons must be spliced precisely, removing the intervening introns, to form meaningful mature messenger RNAs (mRNA) that are transported to the cytoplasm and translated by the ribosomal machinery. To add yet another level of complexity, a process known as alternative splicing exists, whereby a single pre-mRNA can give rise to two or more mature mRNAs depending on the combination of exons spliced together. Alternative splicing of pre-mRNAs is emerging as an important mechanism for gene regulation in many organisms. The classic example of splicing as a regulator of genetic information during a developmental process is sex determination in Drosophila. The now well-characterized cascade of sex-specific alternative splicing events demonstrates nicely how the control of splice site selection during pre-mRNA processing can have a profound effect on the development of an organism. The factors involved in pre-mRNA splicing and alternative splice site selection have been the subject of active study in recent years. Emerging from these studies is a picture of regulation based on protein-protein, protein-RNA, and RNA-RNA interactions. How the interaction of the various splicing constituents is controlled, however, is still poorly understood. One of the mechanisms of regulation that has received attention recently is that of posttranslational phosphorylation. In the following article, we cite the evidence for a role of phosphorylation in constitutive and alternative splicing and discuss some of the recent information on the biochemistry and biology of the enzymes involved.Key words: phosphorylation, splicing, spliceosome, Clk kinases, SR proteins.

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