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

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.

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Altered levels of the Drosophila HRB87F/hrp36 hnRNP protein have limited effects on alternative splicing in vivo.

Molecular Biology of the Cell ◽

10.1091/mbc.7.7.1059 ◽

1996 ◽

Vol 7 (7) ◽

pp. 1059-1073 ◽

Cited By ~ 21

Author(s):

K Zu ◽

M L Sikes ◽

S R Haynes ◽

A L Beyer

Keyword(s):

Alternative Splicing ◽

Splice Site ◽

Site Selection ◽

Exon Skipping ◽

Sr Proteins ◽

Splice Site Selection ◽

General Role ◽

Hnrnp Proteins ◽

Hnrnp Protein

The Drosophila melanogaster genes Hrb87F and Hrb98DE encode the fly proteins HRB87F and HRB98DE (also known as hrp36 and hrp38, respectively) that are most similar in sequence and function to mammalian A/B-type hnRNP proteins. Using overexpression and deletion mutants of Hrb87F, we have tested the hypothesis that the ratio of A/B hnRNP proteins to SR family proteins modulates certain types of alternative splice-site selection. In flies in which HRB87F/hrp36 had been overexpressed 10- to 15-fold above normal levels, aberrant internal exon skipping was induced in at least one endogenous transcript, the dopa decarboxylase (Ddc) pre-mRNA, which previously had been shown to be similarly affected by excess HRB98DE/hrp38. In a second endogenous pre-mRNA, excess HRB87F/hrp36 had no effect on alternative 3' splice-site selection, as expected from mammalian hnRNP studies. Immunolocalization of the excess hnRNP protein showed that it localized correctly to the nucleus, specifically to sites on or near chromosomes, and that the peak of exon-skipping activity in Ddc RNA correlated with the peak of chromosomally associated hnRNP protein. The chromosomal association and level of the SR family of proteins were not significantly affected by the large increase in hnRNP proteins during this time period. Although these results are consistent with a possible role for hnRNP proteins in alternative splicing, the more interesting finding was the failure to detect significant adverse effects on flies with a greatly distorted ratio of hnRNPs to SR proteins. Electron microscopic visualization of the general population of active genes in flies overexpressing hnRNP proteins also indicated that the great majority of genes seemed normal in terms of cotranscriptional RNA processing events, although there were a few abnormalities consistent with rare exon-skipping events. Furthermore, in a Hrb87F null mutant, which is viable, the normal pattern of Ddc alternative splicing was observed, indicating that HRB87F/hrp36 is not required for Ddc splicing regulation. Thus, although splice-site selection can be affected in at least a few genes by gross overexpression of this hnRNP protein, the combined evidence suggests that if it plays a general role in alternative splicing in vivo, the role can be provided by other proteins with redundant functions, and the role is independent of its concentration relative to SR proteins.

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An element in the 5' common exon of the NCAM alternative splicing unit interacts with SR proteins and modulates 5' splice site selection

Nucleic Acids Research ◽

10.1093/nar/27.12.2529 ◽

1999 ◽

Vol 27 (12) ◽

pp. 2529-2537 ◽

Cited By ~ 20

Author(s):

J Cote

Keyword(s):

Alternative Splicing ◽

Splice Site ◽

Site Selection ◽

Sr Proteins ◽

Splice Site Selection

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Role of the 3′ Splice Site in U12-Dependent Intron Splicing

Molecular and Cellular Biology ◽

10.1128/mcb.21.6.1942-1952.2001 ◽

2001 ◽

Vol 21 (6) ◽

pp. 1942-1952 ◽

Cited By ~ 18

Author(s):

Rosemary C. Dietrich ◽

Marian J. Peris ◽

Andrew S. Seyboldt ◽

Richard A. Padgett

Keyword(s):

Splice Site ◽

Site Selection ◽

Intron Splicing ◽

Splice Sites ◽

Splice Site Selection ◽

Branch Site ◽

Site Function

ABSTRACT U12-dependent introns containing alterations of the 3′ splice site AC dinucleotide or alterations in the spacing between the branch site and the 3′ splice site were examined for their effects on splice site selection in vivo and in vitro. Using an intron with a 5′ splice site AU dinucleotide, any nucleotide could serve as the 3′-terminal nucleotide, although a C residue was most active, while a U residue was least active. The penultimate A residue, by contrast, was essential for 3′ splice site function. A branch site-to-3′ splice site spacing of less than 10 or more than 20 nucleotides strongly activated alternative 3′ splice sites. A strong preference for a spacing of about 12 nucleotides was observed. The combined in vivo and in vitro results suggest that the branch site is recognized in the absence of an active 3′ splice site but that formation of the prespliceosomal complex A requires an active 3′ splice site. Furthermore, the U12-type spliceosome appears to be unable to scan for a distal 3′ splice site.

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SR protein kinases: the splice of life

Biochemistry and Cell Biology ◽

10.1139/o99-046 ◽

1999 ◽

Vol 77 (4) ◽

pp. 293-298 ◽

Cited By ~ 54

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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The anti-angiogenic isoforms of VEGF in health and disease

Biochemical Society Transactions ◽

10.1042/bst0371207 ◽

2009 ◽

Vol 37 (6) ◽

pp. 1207-1213 ◽

Cited By ~ 76

Author(s):

Yan Qiu ◽

Coralie Hoareau-Aveilla ◽

Sebastian Oltean ◽

Steven J. Harper ◽

David O. Bates

Keyword(s):

Splice Site ◽

Site Selection ◽

Age Related Macular Degeneration ◽

Splicing Factor ◽

Splice Site Selection ◽

Age Related ◽

Normal Human ◽

Radical Revision

Anti-angiogenic VEGF (vascular endothelial growth factor) isoforms, generated from differential splicing of exon 8, are widely expressed in normal human tissues but down-regulated in cancers and other pathologies associated with abnormal angiogenesis (cancer, diabetic retinopathy, retinal vein occlusion, the Denys–Drash syndrome and pre-eclampsia). Administration of recombinant VEGF165b inhibits ocular angiogenesis in mouse models of retinopathy and age-related macular degeneration, and colorectal carcinoma and metastatic melanoma. Splicing factors and their regulatory molecules alter splice site selection, such that cells can switch from the anti-angiogenic VEGFxxxb isoforms to the pro-angiogenic VEGFxxx isoforms, including SRp55 (serine/arginine protein 55), ASF/SF2 (alternative splicing factor/splicing factor 2) and SRPK (serine arginine domain protein kinase), and inhibitors of these molecules can inhibit angiogenesis in the eye, and splice site selection in cancer cells, opening up the possibility of using splicing factor inhibitors as novel anti-angiogenic therapeutics. Endogenous anti-angiogenic VEGFxxxb isoforms are cytoprotective for endothelial, epithelial and neuronal cells in vitro and in vivo, suggesting both an improved safety profile and an explanation for unpredicted anti-VEGF side effects. In summary, C-terminal distal splicing is a key component of VEGF biology, overlooked by the vast majority of publications in the field, and these findings require a radical revision of our understanding of VEGF biology in normal human physiology.

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Functional properties of p54, a novel SR protein active in constitutive and alternative splicing.

Molecular and Cellular Biology ◽

10.1128/mcb.16.10.5400 ◽

1996 ◽

Vol 16 (10) ◽

pp. 5400-5408 ◽

Cited By ~ 53

Author(s):

W J Zhang ◽

J Y Wu

Keyword(s):

Alternative Splicing ◽

Splice Site ◽

Sr Proteins ◽

Dependent Manner ◽

Sr Protein ◽

Protein Protein Interaction ◽

Small Nuclear Ribonucleoprotein ◽

S100 Extract ◽

U2 Auxiliary Factor ◽

Auxiliary Factor

The p54 protein was previously identified by its reactivity with an autoantiserum. We report here that p54 is a new member of the SR family of splicing factors, as judged from its structural, antigenic, and functional characteristics. Consistent with its identification as an SR protein, p54 can function as a constitutive splicing factor in complementing splicing-deficient HeLa cell S100 extract. However, p54 also shows properties distinct from those of other SR family members, p54 can directly interact with the 65-kDa subunit of U2 auxiliary factor (U2AF65), a protein associated with the 3' splice site. In addition, p54 interacts with other SR proteins but does not interact with the U1 small nuclear ribonucleoprotein U1-70K or the 35-kDa subunit of U2 auxiliary factor (U2AF35). This protein-protein interaction profile is different from those of prototypical SR proteins SC35 and ASF/SF2, both of which interact with U1-70K and U2AF35 but not with U2AF65. p54 promotes the use of the distal 5' splice site in E1A pre-mRNA alternative splicing, while the same site is suppressed by ASF/SF2 and SC35. These findings and the differential tissue distribution of p54 suggest that this novel SR protein may participate in regulation of alternative splicing in a tissue- and substrate-dependent manner.

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Splice site selection and role of the lariat in a group II intron

Journal of Molecular Biology ◽

10.1016/0022-2836(91)90183-7 ◽

1991 ◽

Vol 219 (3) ◽

pp. 415-428 ◽

Cited By ~ 38

Author(s):

Alain Jacquier ◽

Nathalie Jacquesson-Breuleux

Keyword(s):

Splice Site ◽

Site Selection ◽

Group Ii Intron ◽

Splice Site Selection ◽

Group Ii

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Novel Splicing Factor RBM25 Modulates Bcl-x Pre-mRNA 5′ Splice Site Selection

Molecular and Cellular Biology ◽

10.1128/mcb.00560-08 ◽

2008 ◽

Vol 28 (19) ◽

pp. 5924-5936 ◽

Cited By ~ 73

Author(s):

AnYu Zhou ◽

Alexander C. Ou ◽

Aeri Cho ◽

Edward J. Benz ◽

Shu-Ching Huang

Keyword(s):

Splice Site ◽

Dependent Manner ◽

Splice Site Selection ◽

Exon 2 ◽

Small Nuclear Ribonucleoprotein ◽

U1 Snrnp ◽

Nuclear Ribonucleoprotein ◽

Dose Dependent ◽

Selection Of

ABSTRACT RBM25 has been shown to associate with splicing cofactors SRm160/300 and assembled splicing complexes, but little is known about its splicing regulation. Here, we characterize the functional role of RBM25 in alternative pre-mRNA splicing. Increased RBM25 expression correlated with increased apoptosis and specifically affected the expression of Bcl-x isoforms. RBM25 stimulated proapoptotic Bcl-xS 5′ splice site (5′ ss) selection in a dose-dependent manner, whereas its depletion caused the accumulation of antiapoptotic Bcl-xL. Furthermore, RBM25 specifically bound to Bcl-x RNA through a CGGGCA sequence located within exon 2. Mutation in this element abolished the ability of RBM25 to enhance Bcl-xS 5′ ss selection, leading to decreased Bcl-xS isoform expression. Binding of RBM25 was shown to promote the recruitment of the U1 small nuclear ribonucleoprotein particle (snRNP) to the weak 5′ ss; however, it was not required when a strong consensus 5′ ss was present. In support of a role for RBM25 in modulating the selection of a 5′ ss, we demonstrated that RBM25 associated selectively with the human homolog of yeast U1 snRNP-associated factor hLuc7A. These data suggest a novel mode for Bcl-xS 5′ ss activation in which binding of RBM25 with exonic element CGGGCA may stabilize the pre-mRNA-U1 snRNP through interactions with hLuc7A.

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