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 Pyrimidine tracts between the 5' splice site and branch point facilitate splicing and recognition of a small Drosophila intron.

1997 ◽  
Vol 17 (5) ◽  
pp. 2774-2780 ◽  
Author(s):  
C F Kennedy ◽  
S M Berget
Keyword(s):  
Drosophila Melanogaster ◽  
Branch Point ◽  
Splice Site ◽  
Minimum Size ◽  
Cross Linking ◽  
In Vitro Splicing ◽  
Precursor Rna ◽  
Minimal Region

The minimum size for splicing of a vertebrate intron is approximately 70 nucleotides. In Drosophila melanogaster, more than half of the introns are significantly below this minimum yet function well. Such short introns often lack the pyrimidine tract located between the branch point and 3' splice site common to metazoan introns. To investigate if small introns contain special sequences that facilitate their recognition, the sequences and factors required for the splicing of a 59-nucleotide intron from the D. melanogaster mle gene have been examined. This intron contains only a minimal region of interrupted pyrimidines downstream of the branch point. Instead, two longer, uninterrupted C-rich tracts are located between the 5' splice site and branch point. Both of these sequences are required for maximal in vivo and in vitro splicing. The upstream sequences are also required for maximal binding of factors to the 5' splice site, cross-linking of U2AF to precursor RNA, and assembly of the active spliceosome, suggesting that sequences upstream of the branch point influence events at both ends of the small mle intron. Thus, a very short intron lacking a classical pyrimidine tract between the branch point and 3' splice site requires accessory pyrimidine sequences in the short region between the 5' splice site and branch point.

scholarly journals p68 RNA Helicase Is an Essential Human Splicing Factor That Acts at the U1 snRNA-5′ Splice Site Duplex

2002 ◽  
Vol 22 (15) ◽  
pp. 5443-5450 ◽  
Author(s):  
Zhi-Ren Liu
Keyword(s):  
Splice Site ◽  
Early Stage ◽  
Rna Helicase ◽  
Mrna Splicing ◽  
Cross Linking ◽  
Spliceosome Assembly ◽  
U1 Snrna ◽  
U1 Snrnp ◽  
P68 Rna Helicase

ABSTRACT Modulation of the interaction between U1 snRNP and the 5′ splice site (5′ss) is a key event that governs 5′ss recognition and spliceosome assembly. Using the methylene blue-mediated cross-linking method (Z. R. Liu, A. M. Wilkie, M. J. Clemens, and C. W. Smith, RNA 2:611-621, 1996), a 65-kDa protein (p65) was shown to interact with the U1-5′ss duplex during spliceosome assembly (Z. R. Liu, B. Sargueil, and C. W. Smith, Mol. Cell. Biol. 18:6910-6920, 1998). In this report, p65 was identified as p68 RNA helicase and shown to be essential for in vitro pre-mRNA splicing. Depletion of endogenous p68 RNA helicase does not affect the loading of the U1 snRNP to the 5′ss during early stage of splicing. However, dissociation of the U1 from the 5′ss is largely inhibited. The data suggest that p68 RNA helicase functions in destabilizing the U1-5′ss interactions. Furthermore, depletion of p68 RNA helicase arrested spliceosome assembly at the prespliceosome stage, suggesting that p68 may play a role in the transition from prespliceosome to spliceosome.

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 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.

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.

scholarly journals An Upstream AG Determines Whether a Downstream AG Is Selected during Catalytic Step II of Splicing

2001 ◽  
Vol 21 (5) ◽  
pp. 1509-1514 ◽  
Author(s):  
Katrin Chua ◽  
Robin Reed
Keyword(s):  
Branch Point ◽  
Splice Site ◽  
Optimal Location ◽  
Transesterification Reaction ◽  
Precise Localization ◽  
Splice Sites ◽  
Precise Location ◽  
Branch Point Sequence ◽  
General Localization

ABSTRACT Specific mechanisms must exist to ensure fidelity in selecting the AG dinucleotide that functions as the 3′ splice site during the second transesterification step of splicing. Here we show that the optimal location for this AG is within a narrow distance (19 to 23 nucleotides [nt]) downstream from the branch point sequence (BPS). Contrary to previous expectations, AGs located less than 23 nt from the BPS are always recognized, even when a second AG located more optimally downstream is used in the transesterification reaction. Indeed, the AG closest to the BPS actually dictates the precise location of the AG that engages in the reaction. This mechanism, in which the AG is identified by a general localization step followed by a precise localization step, may be used to achieve fidelity while allowing flexibility in the location of 3′ splice sites.

Large-cluster and combined fluorescent and gold immunoprobes

1996 ◽  
Vol 54 ◽  
pp. 892-893
Author(s):  
Richard D. Powell ◽  
James F. Hainfeld ◽  
Carol M. R. Halsey ◽  
David L. Spector ◽  
Shelley Kaurin ◽  
...  
Keyword(s):  
Metal Cluster ◽  
Sulfonyl Chloride ◽  
Gel Filtration ◽  
Cross Linking ◽  
Site Specific ◽  
Fab Fragments ◽  
Cluster Label ◽  
Covalently Linked ◽  
Texas Red ◽  
Fold Excess

Two new types of covalently linked, site-specific immunoprobes have been prepared using metal cluster labels, and used to stain components of cells. Combined fluorescein and 1.4 nm “Nanogold” labels were prepared by using the fluorescein-conjugated tris (aryl) phosphine ligand and the amino-substituted ligand in the synthesis of the Nanogold cluster. This cluster label was activated by reaction with a 60-fold excess of (sulfo-Succinimidyl-4-N-maleiniido-cyclohexane-l-carboxylate (sulfo-SMCC) at pH 7.5, separated from excess cross-linking reagent by gel filtration, and mixed in ten-fold excess with Goat Fab’ fragments against mouse IgG (obtained by reduction of F(ab’)2 fragments with 50 mM mercaptoethylamine hydrochloride). Labeled Fab’ fragments were isolated by gel filtration HPLC (Superose-12, Pharmacia). A combined Nanogold and Texas Red label was also prepared, using a Nanogold cluster derivatized with both and its protected analog: the cluster was reacted with an eight-fold excess of Texas Red sulfonyl chloride at pH 9.0, separated from excess Texas Red by gel filtration, then deprotected with HC1 in methanol to yield the amino-substituted label.

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