Interaction between the Spliceosomal Pre-mRNA Branch Site and U2 snRNP Protein p14

Biochemistry ◽  
2016 ◽  
Vol 55 (4) ◽  
pp. 629-632 ◽  
Author(s):  
William Perea ◽  
Kersten T. Schroeder ◽  
Amy N. Bryant ◽  
Nancy L. Greenbaum
Keyword(s):  
Branch Site ◽  
U2 Snrnp ◽  
Snrnp Protein

scholarly journals A Potential Role for U2AF-SAP 155 Interactions in Recruiting U2 snRNP to the Branch Site

1998 ◽  
Vol 18 (8) ◽  
pp. 4752-4760 ◽  
Author(s):  
Or Gozani ◽  
Judith Potashkin ◽  
Robin Reed
Keyword(s):  
Potential Role ◽  
Specific Binding ◽  
Cross Linking ◽  
Protein Protein Interaction ◽  
Branch Site ◽  
U2 Snrnp ◽  
Snrnp Protein ◽  
Cross Links ◽  
Functional Analyses ◽  
Branchpoint Sequence

ABSTRACT Base pairing between U2 snRNA and the branchpoint sequence (BPS) is essential for pre-mRNA splicing. Because the metazoan BPS is short and highly degenerate, this interaction alone is insufficient for specific binding of U2 snRNP. The splicing factor U2AF binds to the pyrimidine tract at the 3′ splice site in the earliest spliceosomal complex, E, and is essential for U2 snRNP binding in the spliceosomal complex A. We show that the U2 snRNP protein SAP 155 UV cross-links to pre-mRNA on both sides of the BPS in the A complex. SAP 155’s downstream cross-linking site is immediately adjacent to the U2AF binding site, and the two proteins interact directly in protein-protein interaction assays. Using UV cross-linking, together with functional analyses of pre-mRNAs containing duplicated BPSs, we show a direct correlation between BPS selection and UV cross-linking of SAP 155 on both sides of the BPS. Together, our data are consistent with a model in which U2AF binds to the pyrimidine tract in the E complex and then interacts with SAP 155 to recruit U2 snRNP to the BPS.

Differential block of U small nuclear ribonucleoprotein particle interactions during in vitro splicing of adenovirus E1A transcripts containing abnormally short introns

1991 ◽  
Vol 11 (3) ◽  
pp. 1258-1269
Author(s):  
M Himmelspach ◽  
R Gattoni ◽  
C Gerst ◽  
K Chebli ◽  
J Stévenin
Keyword(s):  
Donor Site ◽  
Ribonucleoprotein Particle ◽  
Adenovirus E1a ◽  
Branch Site ◽  
Small Nuclear Ribonucleoprotein ◽  
U2 Snrnp ◽  
U1 Snrnp ◽  
The Common ◽  
Nuclear Ribonucleoprotein

We have studied the consequences of decreasing the donor site-branch site distance on splicing factor-splice site interactions by analyzing alternative splicing of adenovirus E1A pre-mRNAs in vitro. We show that the proximal 13S donor site has a cis-inhibiting effect on the 9S and 12S mRNA reactions when it is brought too close to the common branch site, suggesting that the factor interactions in the common 3' part of the intron are impaired by the U1 small nuclear ribonucleoprotein particle (snRNP) binding to the displaced 13S donor site. Further analysis of the interactions was carried out by studying complex assembly and the accessibility to micrococcal nuclease digestion of 5'-truncated E1A substrates containing only splice sites for the 13S mRNA reaction. A deletion which brings the donor site- branch site distance to 49 nucleotides, which is just below the minimal functional distance, results in a complete block of the U4-U5-U6 snRNP binding, whereas a deletion 15 nucleotides larger results in a severe inhibition of the formation of the U2 snRNP-containing complexes. Sequence accessibility analyses performed by using the last mini-intron-containing transcript demonstrate that the interactions of U2 snRNP with the branch site are strongly impaired whereas the initial bindings of U1 snRNP to the donor site and of specific factors to the 3' splice site are not significantly modified. Our results strongly suggest that the interaction of U1 snRNP with the donor site of a mini-intron is stable enough in vitro to affect the succession of events leading to U2 snRNP binding with the branch site.

scholarly journals The ATP requirement for U2 snRNP addition is linked to the pre-mRNA region 5′ to the branch site

RNA ◽  
2001 ◽  
Vol 7 (9) ◽  
pp. 1298-1309 ◽  
Author(s):  
CATHERINE M. NEWNHAM ◽  
CHARLES C. QUERY
Keyword(s):  
Branch Site ◽  
U2 Snrnp

scholarly journals Small nuclear ribonucleoprotein (RNP) U2 contains numerous additional proteins and has a bipartite RNP structure under splicing conditions.

1993 ◽  
Vol 13 (1) ◽  
pp. 307-319 ◽  
Author(s):  
S E Behrens ◽  
K Tyc ◽  
B Kastner ◽  
J Reichelt ◽  
R Lührmann
Keyword(s):  
Electron Microscope ◽  
Salt Concentration ◽  
Rnase H ◽  
Globular Domain ◽  
Branch Site ◽  
Small Nuclear Ribonucleoprotein ◽  
U2 Snrnp ◽  
Specific Proteins ◽  
Molecular Masses ◽  
Nuclear Ribonucleoprotein

Small nuclear (sn) ribonucleoprotein (RNP) U2 functions in the splicing of mRNA by recognizing the branch site of the unspliced pre-mRNA. When HeLa nuclear splicing extracts are centrifuged on glycerol gradients, U2 snRNPs sediment at either 12S (under high salt concentration conditions) or 17S (under low salt concentration conditions). We isolated the 17S U2 snRNPs from splicing extracts under nondenaturing conditions by using centrifugation and immunoaffinity chromatography and examined their structure by electron microscope. In addition to common proteins B', B, D1, D2, D3, E, F, and G and U2-specific proteins A' and B", which are present in the 12S U2 snRNP, at least nine previously unidentified proteins with apparent molecular masses of 35, 53, 60, 66, 92, 110, 120, 150, and 160 kDa bound to the 17S U2 snRNP. The latter proteins dissociate from the U2 snRNP at salt concentrations above 200 mM, yielding the 12S U2 snRNP particle. Under the electron microscope, the 17S U2 snRNPs exhibited a bipartite appearance, with two main globular domains connected by a short filamentous structure that is sensitive to RNase. These findings suggest that the additional globular domain, which is absent from 12S U2 snRNPs, contains some of the 17S U2-specific proteins. The 5' end of the RNA in the U2 snRNP is more exposed for reaction with RNase H and with chemical probes when the U2 snRNP is in the 17S form than when it is in the 12S form. Removal of the 5' end of this RNA reduces the snRNP's Svedberg value from 17S to 12S. Along with the peculiar morphology of the 17S snRNP, these data indicate that most of the 17S U2-specific proteins are bound to the 5' half of the U2 snRNA.

scholarly journals Accumulation of a novel spliceosomal complex on pre-mRNAs containing branch site mutations.

1995 ◽  
Vol 15 (10) ◽  
pp. 5750-5756 ◽  
Author(s):  
P Champion-Arnaud ◽  
O Gozani ◽  
L Palandjian ◽  
R Reed
Keyword(s):  
Second Step ◽  
Apparent Effect ◽  
Ribonucleoprotein Particle ◽  
Branch Site ◽  
Small Nuclear Ribonucleoprotein ◽  
U2 Snrnp ◽  
U1 Snrnp ◽  
Nuclear Ribonucleoprotein ◽  
Branchpoint Sequence ◽  
Independent Reaction

Pre-mRNA assembles into spliceosomal complexes in the stepwise pathway E-->A-->B-->C. We show that mutations in the metazoan branchpoint sequence (BPS) have no apparent effect on E complex formation but block the assembly of the A complex and the UV cross-linking of U2 small nuclear ribonucleoprotein particle (snRNP) proteins. Unexpectedly, a novel complex, designated E*, assembles on pre-mRNAs containing BPS mutations. Unlike the E complex, the E* complex accumulates in the presence of ATP. U1 snRNP and U2AF, which are tightly bound to pre-mRNA in the E complex, are not tightly bound in the E* complex. Significantly, previous work showed that U1 snRNP and U2AF become destabilized from pre-mRNA after E complex assembly on normal pre-mRNAs. Thus, our data are consistent with a model in which there are two steps in the transition from the E complex to the A complex (E-->E*-->A). In the first step, U1 snRNP and U2AF are destabilized in an ATP-dependent, BPS-independent reaction. In the second step, the stable binding of U2 snRNP occurs in a BPS-dependent reaction.

scholarly journals A minimal spliceosomal complex A recognizes the branch site and polypyrimidine tract.

1997 ◽  
Vol 17 (5) ◽  
pp. 2944-2953 ◽  
Author(s):  
C C Query ◽  
P S McCaw ◽  
P A Sharp
Keyword(s):  
Specific Interactions ◽  
Polypyrimidine Tract ◽  
Detailed Understanding ◽  
Branch Site ◽  
U2 Snrnp ◽  
Dependent Activity ◽  
U1 Snrnp ◽  
Polypyrimidine Tract Binding Proteins ◽  
Per Se ◽  
Rna Complex

The association of U2 snRNP with the pre-mRNA branch region is a critical step in the assembly of spliceosomal complexes. We describe an assembly process that reveals both minimal requirements for formation of a U2 snRNP-substrate RNA complex, here designated the Amin complex, and specific interactions with the branch site adenosine. The substrate is a minimal RNA oligonucleotide, containing only a branch sequence and polypyrimidine tract. Interactions at the branch site adenosine and requirements for polypyrimidine tract-binding proteins for the Amin complex are the same as those of authentic prespliceosome complex A. Surprisingly, Amin complex formation does not require U1 snRNP or ATP, suggesting that these factors are not necessary for stable binding of U2 snRNP per se, but rather are necessary for accessibility of components on longer RNA substrates. Furthermore, there is an ATP-dependent activity that releases or destabilizes U2 snRNP from branch sequences. The simplicity of the Amin complex will facilitate a detailed understanding of the assembly of prespliceosomes.

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