scholarly journals Brr2 is a splicing fidelity factor

2018 ◽  
Author(s):  
Megan Mayerle ◽  
Christine Guthrie
Keyword(s):  
Site Selection ◽  
Site Fidelity ◽  
Rna Binding ◽  
Intron Retention ◽  
Mrna Splicing ◽  
Splice Site Selection ◽  
Systematic Analysis ◽  
Branch Site ◽  
Spliceosome Activation

AbstractMany spliceosomal DExD/H box helicases act as fidelity factors during pre-mRNA splicing, promoting on-pathway interactions while simultaneously minimizing errors. Mutations linked to Retinitis Pigmentosa (RP), a form of heritable blindness, map to key domains of spliceosomal helicase Brr2 (SNRNP200 in humans). Previous data show that such mutations negatively impact spliceosome activation, likely due to defects in brr2-RP RNA binding, helicase, and ATPase activities. Furthermore, data from human reporter constructs suggest that brr2-RP might impact 5′ splice site selection. Here we undertake a systematic analysis of brr2-RP effects on splicing fidelity. We show that a subset of brr2-RP mutants exhibit intron retention in vivo. Furthermore, brr2-RP mutants display hyperaccurate and/or error-prone splicing of a variety of splicing reporters. Branch-site fidelity is particularly impacted in this reporter assay. In addition, multiple brr2-RP alleles genetically interact with prp16 alleles known to impact the fidelity of branch site selection. Together these data implicate Brr2 in the fidelity of branch-site selection, and suggest that RP results not just from defects in spliceosome activation, but also from fidelity defects arising throughout the splicing cycle and in splicing fidelity.

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

2001 ◽  
Vol 21 (6) ◽  
pp. 1942-1952 ◽  
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.

scholarly journals The anti-angiogenic isoforms of VEGF in health and disease

2009 ◽  
Vol 37 (6) ◽  
pp. 1207-1213 ◽  
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.

scholarly journals Overexpression of Essential Splicing Factor ASF/SF2 Blocks the Temporal Shift in Adenovirus Pre-mRNA Splicing and Reduces Virus Progeny Formation

2000 ◽  
Vol 74 (19) ◽  
pp. 9002-9009 ◽  
Author(s):  
Magnus Molin ◽  
Göran Akusjärvi
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Recombinant Adenovirus ◽  
Mrna Splicing ◽  
General Tendency ◽  
Viral Control ◽  
Splice Site Selection ◽  
Sr Protein ◽  
Virus Growth ◽  
Temporal Shift

ABSTRACT Expression of cytoplasmic mRNA from most adenovirus transcription units is subjected to a temporal regulation at the level of alternative pre-mRNA splicing. The general tendency is that splice site selection changes from proximal to distal late after infection. Interestingly, ASF/SF2, which is a prototypical member of the SR family of splicing factors, has the opposite effect on splice site selection, inducing an increase in proximal splice site usage. We have previously shown that SR proteins late during an adenovirus infection become partially inactivated as splicing regulatory proteins. A prediction from these results is that overexpression of an SR protein, such as ASF/SF2, during virus growth will interfere with virus replication by disturbing the balance of functional and nonfunctional ASF/SF2 in the infected cell. To test this hypothesis, we reconstructed a recombinant adenovirus expressing ASF/SF2 under the transcriptional control of a regulated promoter. The results show that, as predicted, induction of ASF/SF2 during lytic virus growth prevents the early to late shift in mRNA expression from both early (E1A and E1B) and late (L1) transcription units. Furthermore, ASF/SF2 overexpression blocks viral DNA replication and reduces selectively cytoplasmic accumulation of major late mRNA, resulting in a lower virus yield. Collectively, our results provide additional support for the hypothesis that viral control of SR protein function is important for the proper expression of viral proteins during lytic virus growth.

scholarly journals Promoter Choice Determines Splice Site Selection in Protocadherin α and γ Pre-mRNA Splicing

2002 ◽  
Vol 10 (1) ◽  
pp. 21-33 ◽  
Author(s):  
Bosiljka Tasic ◽  
Christoph E. Nabholz ◽  
Kristin K. Baldwin ◽  
Youngwook Kim ◽  
Erroll H. Rueckert ◽  
...  
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Mrna Splicing ◽  
Splice Site Selection

scholarly journals The A1 and A1B proteins of heterogeneous nuclear ribonucleoparticles modulate 5' splice site selection in vivo.

1994 ◽  
Vol 91 (15) ◽  
pp. 6924-6928 ◽  
Author(s):  
X. Yang ◽  
M. R. Bani ◽  
S. J. Lu ◽  
S. Rowan ◽  
Y. Ben-David ◽  
...  
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Splice Site Selection

scholarly journals Exonic Sequences in the 5′ Untranslated Region of α-Tubulin mRNA Modulate trans Splicing inTrypanosoma brucei

1998 ◽  
Vol 18 (8) ◽  
pp. 4620-4628 ◽  
Author(s):  
Carlos López-Estraño ◽  
Christian Tschudi ◽  
Elisabetta Ullu
Keyword(s):  
Splice Site ◽  
Competition Assay ◽  
Splice Site Selection ◽  
Tubulin Gene ◽  
Branch Site ◽  
Trans Splicing ◽  
Tubulin Mrna ◽  
Splicing Efficiency ◽  
Β Tubulin

ABSTRACT Previous studies have identified a conserved AG dinucleotide at the 3′ splice site (3′SS) and a polypyrimidine (pPy) tract that are required for trans splicing of polycistronic pre-mRNAs in trypanosomatids. Furthermore, the pPy tract of the Trypanosoma brucei α-tubulin 3′SS region is required to specify accurate 3′-end formation of the upstream β-tubulin gene and transsplicing of the downstream α-tubulin gene. Here, we employed an in vivo cis competition assay to determine whether sequences other than those of the AG dinucleotide and the pPy tract were required for 3′SS identification. Our results indicate that a minimal α-tubulin 3′SS, from the putative branch site region to the AG dinucleotide, is not sufficient for recognition by thetrans-splicing machinery and that polyadenylation is strictly dependent on downstream trans splicing. We show that efficient use of the α-tubulin 3′SS is dependent upon the presence of exon sequences. Furthermore, β-tubulin, but not actin exon sequences or unrelated plasmid sequences, can replace α-tubulin exon sequences for accurate trans-splice-site selection. Taken together, these results support a model in which the informational content required for efficient trans splicing of the α-tubulin pre-mRNA includes exon sequences which are involved in modulation of trans-splicing efficiency. Sequences that positively regulate trans splicing might be similar tocis-splicing enhancers described in other systems.

scholarly journals Factors influencing alternative splice site utilization in vivo.

1987 ◽  
Vol 7 (2) ◽  
pp. 738-748 ◽  
Author(s):  
X Y Fu ◽  
J L Manley
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Simian Virus 40 ◽  
Cell Types ◽  
Simian Virus ◽  
Splice Sites ◽  
Splice Site Selection ◽  
Culture Cells ◽  
293 Cells

To study factors that influence the choice of alternative pre-mRNA splicing pathways, we introduced plasmids expressing either wild-type or mutated simian virus 40 (SV40) early regions into tissue culture cells and then measured the quantities of small-t and large-T RNAs produced. One important element controlling splice site selection was found to be the size of the intron removed in the production of small-t mRNA; expansion of this intron (from 66 to 77 or more nucleotides) resulted in a substantial increase in the amount of small-t mRNA produced relative to large-T mRNA. This suggests that in the normal course of SV40 early pre-mRNA processing, large-T splicing is at a competitive advantage relative to small-t splicing because of the small size of the latter intron. Several additional features of the pre-mRNA that can influence splice site selection were also identified by analyzing the effects of mutations containing splice site duplications. These include the strengths of competing 5' splice sites and the relative positions of splice sites in the pre-mRNA. Finally, we showed that the ratio of small-t to large-T mRNA was 10 to 15-fold greater in human 293 cells than in HeLa cells or other mammalian cell types. These results suggest the existence of cell-specific trans-acting factors that can dramatically alter the pattern of splice site selection in a pre-mRNA.

scholarly journals Splicing Control in Normal and Aberrant Erythropoiesis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. SCI-47-SCI-47
Author(s):  
Esther Obeng
Keyword(s):  
Gene Expression ◽  
Rna Processing ◽  
Iron Transport ◽  
Rna Binding ◽  
Intron Retention ◽  
Point Mutations ◽  
Cytoskeletal Proteins ◽  
Mrna Splicing ◽  
Splice Sites ◽  
Erythroid Maturation

Abstract Alternative splicing is employed by all eukaryotic cells to increase proteome diversity and to regulate gene expression. RNA sequencing analysis of purified populations of erythroblasts at different stages of maturation has led to the identification of a dynamic alternative splicing program that directly modulates the protein isoform expression of cytoskeletal proteins and genes involved in RNA processing, heme biosynthesis, and iron transport. Regulated interactions of multiple RNA-binding proteins and cis-regulatory sequences located within exons or their flanking introns promote or inhibit functional spliceosome assembly at splice junctions, leading to altered exon inclusion or intron retention. Exon skipping regulates tissue and stage specific isoform expression of red cell membrane cytoskeletal proteins including EPB41, ankyrin, and band 3. Intron retention can lead to a frame shift during translation and introduction of a premature termination codon (PTC), that marks the transcript for degradation via the nonsense mediated decay pathway (NMD) upon export from the nucleus into the cytoplasm. Intron retention leading to posttranscriptional regulation of gene expression during terminal erythroid maturation has been identified in genes involved in RNA processing and iron transport including SF3B1, SNRNP70, SLC25A37 and SLC25A28. Mutations that alter mRNA splice sites or introduce PTCs lead to a variety of congenital anemias including beta thalassemia, hereditary pyropoikilocytosis, hereditary elliptocytosis, and hereditary spherocytosis. Aberrant mRNA splicing has subsequently been shown to lead to acquired anemias in subsets of patients with myelodysplastic syndromes (MDS). Somatic missense mutations in components of the spliceosome are the most common category of mutations in MDS. These point mutations lead to changes in the RNA binding specificity of the involved proteins and aberrant splicing of a subset of transcripts. Mutant SF3B1, the most commonly mutated splicing factor in MDS, has been shown to cause aberrant pre-mRNA splicing and an increase in transcripts predicted to undergo NMD due to use of upstream, cryptic 3' splice sites. Our group and others evaluating the strong genotype-phenotype association between SF3B1 point mutations and subtypes of MDS with ring sideroblasts have shown that the expression of the mitochondrial iron transporter, ABCB7, is decreased in samples from SF3B1-mutant MDS patients due to cryptic 3' splice site selection and introduction of a PTC between exons 8 and 9. The identification and functional validation of additional aberrantly spliced mutant-SF3B1 target genes is ongoing, with the goal of understanding how point mutations in a core component of the mRNA splicing machinery can lead to such specific effects on erythroid maturation. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document