scholarly journals Proximity of the U12 snRNA with both the 5′ Splice Site and the Branch Point during Early Stages of Spliceosome Assembly

2005 ◽  
Vol 25 (12) ◽  
pp. 4813-4825 ◽  
Author(s):  
Mikko J. Frilander ◽  
Xiaojuan Meng
Keyword(s):  
Branch Point ◽  
Splice Site ◽  
Cross Linking ◽  
Catalytic Core ◽  
Spliceosome Assembly ◽  
Site Specific ◽  
Early Stages ◽  
Branch Point Sequence

ABSTRACT U12 snRNA is required for branch point recognition in the U12-dependent spliceosome. Using site-specific cross-linking, we have captured an unexpected interaction between the 5′ end of the U12 snRNA and the −2 position upstream of the 5′ splice site of P120 and SCN4a splicing substrates. The U12 snRNA nucleotides that contact the 5′ exon are the same ones that form the catalytically important helix Ib with U6atac snRNA in the spliceosome catalytic core. However, the U12/5′ exon interaction is transient, occurring prior to the entry of the U4atac/U6atac.U5 tri-snRNP to the spliceosome. This suggests that the helix Ib region of U12 snRNA is positioned near the 5′ splice site early during spliceosome assembly and only later interacts with U6atac to form helix Ib. We also provide evidence that U12 snRNA can simultaneously interact with 5′ exon sequences near 5′ splice site and the branch point sequence, suggesting that the 5′ splice site and branch point sequences are separated by <40 to 50 Å in the complex A of the U12-dependent spliceosome. Thus, no major rearrangements are subsequently needed to position these sites for the first step of catalysis.

scholarly journals The intronic branch point sequence is under strong evolutionary constraint in the bovine and human genome

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Naveen Kumar Kadri ◽  
Xena Marie Mapel ◽  
Hubert Pausch
Keyword(s):  
Branch Point ◽  
Purifying Selection ◽  
Mrna Splicing ◽  
Evolutionary Constraints ◽  
Evolutionary Constraint ◽  
Functional Importance ◽  
Coding Sequences ◽  
Cis Acting ◽  
Branch Point Sequence ◽  
Human Genomes

AbstractThe branch point sequence is a cis-acting intronic motif required for mRNA splicing. Despite their functional importance, branch point sequences are not routinely annotated. Here we predict branch point sequences in 179,476 bovine introns and investigate their variability using a catalogue of 29.4 million variants detected in 266 cattle genomes. We localize the bovine branch point within a degenerate heptamer “nnyTrAy”. An adenine residue at position 6, that acts as branch point, and a thymine residue at position 4 of the heptamer are more strongly depleted for mutations than coding sequences suggesting extreme purifying selection. We provide evidence that mutations affecting these evolutionarily constrained residues lead to alternative splicing. We confirm evolutionary constraints on branch point sequences using a catalogue of 115 million SNPs established from 3,942 human genomes of the gnomAD database.

scholarly journals U2 snRNA-Protein Contacts in Purified Human 17S U2 snRNPs and in Spliceosomal A and B Complexes

2006 ◽  
Vol 26 (7) ◽  
pp. 2803-2816 ◽  
Author(s):  
Olexandr Dybkov ◽  
Cindy L. Will ◽  
Jochen Deckert ◽  
Nastaran Behzadnia ◽  
Klaus Hartmuth ◽  
...  
Keyword(s):  
Branch Point ◽  
Protein Interactions ◽  
Cross Linking ◽  
Molecular Architecture ◽  
Point Selection ◽  
Stem Loop ◽  
U2 Snrna ◽  
Branch Site ◽  
Branch Point Sequence ◽  
U2 Snrnp

ABSTRACT The 17S U2 snRNP plays an essential role in branch point selection and catalysis during pre-mRNA splicing. Much remains to be learned about the molecular architecture of the U2 snRNP, including which proteins contact the functionally important 5′ end of the U2 snRNA. Here, RNA-protein interactions within immunoaffinity-purified human 17S U2 snRNPs were analyzed by lead(II)-induced RNA cleavage and UV cross-linking. Contacts between the U2 snRNA and SF3a60, SF3b49, SF3b14a/p14 and SmG and SmB were detected. SF3b49 appears to make multiple contacts, interacting with the 5′ end of U2 and nucleotides in loops I and IIb. SF3a60 also contacted different regions of the U2 snRNA, including the base of stem-loop I and a bulge in stem-loop III. Consistent with it contacting the pre-mRNA branch point adenosine, SF3b14a/p14 interacted with the U2 snRNA near the region that base pairs with the branch point sequence. A comparison of U2 cross-linking patterns obtained with 17S U2 snRNP versus purified spliceosomal A and B complexes revealed that RNA-protein interactions with stem-loop I and the branch site-interacting region of U2 are dynamic. These studies provide important insights into the molecular architecture of 17S U2 snRNPs and reveal U2 snRNP remodeling events during spliceosome assembly.

scholarly journals Wobble Splicing Reveals the Role of the Branch Point Sequence-to-NAGNAG Region in 3′ Tandem Splice Site Selection

2007 ◽  
Vol 27 (16) ◽  
pp. 5835-5848 ◽  
Author(s):  
Kuo-Wang Tsai ◽  
Woan-Yuh Tarn ◽  
Wen-chang Lin
Keyword(s):  
Branch Point ◽  
Splice Site ◽  
Site Selection ◽  
Protein Function ◽  
Upstream Region ◽  
Nucleotide Polymorphisms ◽  
Splice Site Selection ◽  
Independent Manner ◽  
Branch Point Sequence

ABSTRACT Alternative splicing involving the 3′ tandem splice site NAGNAG sequence may play a role in the structure-function diversity of proteins. However, how 3′ tandem splice site utilization is determined is not well understood. We previously demonstrated that 3′ NAGNAG-based wobble splicing occurs mostly in a tissue- and developmental stage-independent manner. Bioinformatic analysis reveals that the nucleotide preceding the AG dinucleotide may influence 3′ splice site utilization; this is also supported by an in vivo splicing assay. Moreover, we found that the intron sequence plays an important role in 3′ splice site selection for NAGNAG wobble splicing. Mutations of the region between the branch site and the NAGNAG 3′ splice site, indeed, affected the ratio of the distal/proximal AG selection. Finally, we found that single nucleotide polymorphisms around the NAGNAG motif could affect the splice site choice, which may lead to a change in mRNA patterns and influence protein function. We conclude that the NAGNAG motif and its upstream region to the branch point sequence are required for 3′ tandem splice site selection.

scholarly journals A Novel Intronic cis Element, ISE/ISS-3, Regulates Rat Fibroblast Growth Factor Receptor 2 Splicing through Activation of an Upstream Exon and Repression of a Downstream Exon Containing a Noncanonical Branch Point Sequence

2005 ◽  
Vol 25 (1) ◽  
pp. 250-263 ◽  
Author(s):  
Ruben H. Hovhannisyan ◽  
Russ P. Carstens
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Branch Point ◽  
Fibroblast Growth Factor Receptor ◽  
Growth Factor Receptor ◽  
Primary Branch ◽  
Fibroblast Growth ◽  
Cell Type ◽  
Branch Point Sequence ◽  
Cell Type Specific

ABSTRACT Mutually exclusive splicing of fibroblast growth factor receptor 2 (FGFR2) exons IIIb and IIIc yields two receptor isoforms, FGFR2-IIIb and -IIIc, with distinctly different ligand binding properties. Several RNA cis elements in the intron (intron 8) separating these exons have been described that are required for splicing regulation. Using a heterologous splicing reporter, we have identified a new regulatory element in this intron that confers cell-type-specific inclusion of an unrelated exon that mirrors its ability to promote cell-type-specific inclusion of exon IIIb. This element promoted inclusion of exon IIIb while at the same time silencing exon IIIc inclusion in cells expressing FGFR2-IIIb; hence, we have termed this element ISE/ISS-3 (for “intronic splicing enhancer-intronic splicing silencer 3”). Silencing of exon IIIc splicing by ISE/ISS-3 was shown to require a branch point sequence (BPS) using G as the primary branch nucleotide. Replacing a consensus BPS with A as the primary branch nucleotide resulted in constitutive splicing of exon IIIc. Our results suggest that the branch point sequence constitutes an important component that can contribute to the efficiency of exon definition of alternatively spliced cassette exons. Noncanonical branch points may thus facilitate cell-type-specific silencing of regulated exons by flanking cis elements.

scholarly journals Identification and characterization by antisense oligonucleotides of exon and intron sequences required for splicing.

1994 ◽  
Vol 14 (11) ◽  
pp. 7445-7454 ◽  
Author(s):  
Z Dominski ◽  
R Kole
Keyword(s):  
Branch Point ◽  
Splice Site ◽  
Consensus Sequence ◽  
Aberrant Splice ◽  
Splice Sites ◽  
Branch Point Sequence ◽  
Splice Site Sequence ◽  
Internal Exon ◽  
Cryptic Splice Sites

Certain thalassemic human beta-globin pre-mRNAs carry mutations that generate aberrant splice sites and/or activate cryptic splice sites, providing a convenient and clinically relevant system to study splice site selection. Antisense 2'-O-methyl oligoribonucleotides were used to block a number of sequences in these pre-mRNAs and were tested for their ability to inhibit splicing in vitro or to affect the ratio between aberrantly and correctly spliced products. By this approach, it was found that (i) up to 19 nucleotides upstream from the branch point adenosine are involved in proper recognition and functioning of the branch point sequence; (ii) whereas at least 25 nucleotides of exon sequences at both 3' and 5' ends are required for splicing, this requirement does not extend past the 5' splice site sequence of the intron; and (iii) improving the 5' splice site of the internal exon to match the consensus sequence strongly decreases the accessibility of the upstream 3' splice site to antisense 2'-O-methyl oligoribonucleotides. This result most likely reflects changes in the strength of interactions near the 3' splice site in response to improvement of the 5' splice site and further supports the existence of communication between these sites across the exon.

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