scholarly journals U1 small nuclear ribonucleoprotein particle-specific proteins interact with the first and second stem-loops of U1 RNA, with the A protein binding directly to the RNA independently of the 70K and Sm proteins.

1989 ◽  
Vol 9 (8) ◽  
pp. 3360-3368 ◽  
Author(s):  
J R Patton ◽  
W Habets ◽  
W J van Venrooij ◽  
T Pederson
Keyword(s):  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Ribonucleoprotein Particle ◽  
Small Nuclear Ribonucleoprotein ◽  
Core Proteins ◽  
U1 Snrnp ◽  
Specific Proteins ◽  
C Proteins ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoprotein Particle

The U1 small nuclear ribonucleoprotein particle (U1 snRNP), a cofactor in pre-mRNA splicing, contains three proteins, termed 70K, A, and C, that are not present in the other spliceosome-associated snRNPs. We studied the binding of the A and C proteins to U1 RNA, using a U1 snRNP reconstitution system and an antibody-induced nuclease protection technique. Antibodies that reacted with the A and C proteins induced nuclease protection of the first two stem-loops of U1 RNA in reconstituted U1 snRNP. Detailed analysis of the antibody-induced nuclease protection patterns indicated the existence of relatively long-range protein-protein interactions in the U1 snRNP, with the 5' end of U1 RNA and its associated specific proteins interacting with proteins bound to the Sm domain near the 3' end. UV cross-linking experiments in conjunction with an A-protein-specific antibody demonstrated that the A protein bound directly to the U1 RNA rather than assembling in the U1 snRNP exclusively via protein-protein interactions. This conclusion was supported by additional experiments revealing that the A protein could bind to U1 RNA in the absence of bound 70K and Sm core proteins.

U1 small nuclear ribonucleoprotein particle-specific proteins interact with the first and second stem-loops of U1 RNA, with the A protein binding directly to the RNA independently of the 70K and Sm proteins

1989 ◽  
Vol 9 (8) ◽  
pp. 3360-3368
Author(s):  
J R Patton ◽  
W Habets ◽  
W J van Venrooij ◽  
T Pederson
Keyword(s):  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Ribonucleoprotein Particle ◽  
Small Nuclear Ribonucleoprotein ◽  
Core Proteins ◽  
U1 Snrnp ◽  
Specific Proteins ◽  
C Proteins ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoprotein Particle

The U1 small nuclear ribonucleoprotein particle (U1 snRNP), a cofactor in pre-mRNA splicing, contains three proteins, termed 70K, A, and C, that are not present in the other spliceosome-associated snRNPs. We studied the binding of the A and C proteins to U1 RNA, using a U1 snRNP reconstitution system and an antibody-induced nuclease protection technique. Antibodies that reacted with the A and C proteins induced nuclease protection of the first two stem-loops of U1 RNA in reconstituted U1 snRNP. Detailed analysis of the antibody-induced nuclease protection patterns indicated the existence of relatively long-range protein-protein interactions in the U1 snRNP, with the 5' end of U1 RNA and its associated specific proteins interacting with proteins bound to the Sm domain near the 3' end. UV cross-linking experiments in conjunction with an A-protein-specific antibody demonstrated that the A protein bound directly to the U1 RNA rather than assembling in the U1 snRNP exclusively via protein-protein interactions. This conclusion was supported by additional experiments revealing that the A protein could bind to U1 RNA in the absence of bound 70K and Sm core proteins.

Identification and characterization of the small nuclear ribonucleoprotein particle D' core protein

1990 ◽  
Vol 10 (9) ◽  
pp. 4480-4485
Author(s):  
J Andersen ◽  
R J Feeney ◽  
G W Zieve
Keyword(s):  
Electrophoretic Mobility ◽  
Protein Complexes ◽  
Ribonucleoprotein Particle ◽  
Small Nuclear Ribonucleoprotein ◽  
Core Proteins ◽  
Nuclear Ribonucleoprotein ◽  
Identification And Characterization ◽  
Snrnp Core ◽  
Small Nuclear Ribonucleoprotein Particle

The addition of urea to sodium dodecyl sulfate (SDS)-polyacrylamide gels has allowed the identification and characterization of the small nuclear ribonucleoprotein particle (snRNP) D' protein and has also improved resolution of the E, F, and G snRNP core proteins. In standard SDS-polyacrylamide gels, the D' and D snRNP core proteins comigrate at approximately 16 kilodaltons. The addition of urea to the separating gel caused the D' protein to shift to a slower electrophoretic mobility that is distinct from that of the D protein. The shift to a slower electrophoretic mobility in the presence of urea suggests that the D' protein has extensive secondary structure that is not totally disrupted by SDS alone. Both N-terminal sequencing and partial peptide maps indicate that the D and D' proteins are distinct gene products, and the sequence data have identified the faster moving of the two proteins as the previously cloned D protein (L. A. Rokeach, J. A. Haselby, and S. O. Hoch, Proc. Natl. Acad. Sci. USA 85:4832-4836, 1988). In the cytoplasm, the D protein is found primarily in the small-nuclear-RNA-free 6S protein complexes, while the D' protein is found primarily in the 20S protein complexes. Like the D protein, the D' protein is an autoantigen in patients with systemic lupus erythematosus and is recognized by some of the Sm class of autoimmune antisera.

scholarly journals The Human U5-220kD Protein (hPrp8) Forms a Stable RNA-Free Complex with Several U5-Specific Proteins, Including an RNA Unwindase, a Homologue of Ribosomal Elongation Factor EF-2, and a Novel WD-40 Protein

1998 ◽  
Vol 18 (11) ◽  
pp. 6756-6766 ◽  
Author(s):  
Tilmann Achsel ◽  
Katharina Ahrens ◽  
Hero Brahms ◽  
Stefan Teigelkamp ◽  
Reinhard Lührmann
Keyword(s):  
Conformational Changes ◽  
Protein Interactions ◽  
Elongation Factor ◽  
Sodium Thiocyanate ◽  
Sequence Motifs ◽  
Protein Protein Interactions ◽  
Small Nuclear Ribonucleoprotein ◽  
Core Proteins ◽  
Specific Proteins ◽  
Biochemical Analyses

ABSTRACT The human small nuclear ribonucleoprotein (snRNP) U5 is biochemically the most complex of the snRNP particles, containing not only the Sm core proteins but also 10 particle-specific proteins. Several of these proteins have sequence motifs which suggest that they participate in conformational changes of RNA and protein. Together, the specific proteins comprise 85% of the mass of the U5 snRNP particle. Therefore, protein-protein interactions should be highly important for both the architecture and the function of this particle. We investigated protein-protein interactions using both native and recombinant U5-specific proteins. Native U5 proteins were obtained by dissociation of U5 snRNP particles with the chaotropic salt sodium thiocyanate. A stable, RNA-free complex containing the 116-kDa EF-2 homologue (116kD), the 200kD RNA unwindase, the 220kD protein, which is the orthologue of the yeast Prp8p protein, and the U5-40kD protein was detected by sedimentation analysis of the dissociated proteins. By cDNA cloning, we show that the 40kD protein is a novel WD-40 repeat protein and is thus likely to mediate regulated protein-protein interactions. Additional biochemical analyses demonstrated that the 220kD protein binds simultaneously to the 40- and the 116kD proteins and probably also to the 200kD protein. Since the 220kD protein is also known to contact both the pre-mRNA and the U5 snRNA, it is in a position to relay the functional state of the spliceosome to the other proteins in the complex and thus modulate their activity.

scholarly journals Combinatorial splicing of exon pairs by two-site binding of U1 small nuclear ribonucleoprotein particle.

1991 ◽  
Vol 11 (12) ◽  
pp. 5919-5928 ◽  
Author(s):  
P J Grabowski ◽  
F U Nasim ◽  
H C Kuo ◽  
R Burch
Keyword(s):  
Splice Site ◽  
Failure To Rescue ◽  
Ribonucleoprotein Particle ◽  
Upstream Site ◽  
Site Model ◽  
Small Nuclear Ribonucleoprotein ◽  
U1 Snrnp ◽  
Alternatively Spliced ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoprotein Particle

A two-site model for the binding of U1 small nuclear ribonucleoprotein particle (U1 snRNP) was tested in order to understand how exon partners are selected in complex pre-mRNAs containing alternative exons. In this model, it is proposed that two U1 snRNPs define a functional unit of splicing by base pairing to the 3' boundary of the downstream exon as well as the 5' boundary of the intron to be spliced. Three-exon substrates contained the alternatively spliced exon 4 (E4) region of the preprotachykinin gene. Combined 5' splice site mutations at neighboring exons demonstrate that weakened binding of U1 snRNP at the downstream site and improved U1 snRNP binding at the upstream site result in the failure to rescue splicing of the intron between the mutations. These results indicate the stringency of the requirement for binding a second U1 snRNP to the downstream 5' splice site for these substrates as opposed to an alternative model in which a certain threshold level of U1 snRNP can be provided at either site. Further support for the two-site model is provided by single-site mutations in the 5' splice site of the third exon, E5, that weaken base complementarity to U1 RNA. These mutations block E5 branchpoint formation and, surprisingly, generate novel branchpoints that are specified chiefly by their proximity to a cryptic 5' splice site located at the 3' terminus of the pre-mRNA. The experiments shown here demonstrate a true stimulation of 3' splice site activity by the downstream binding of U1 snRNP and suggest a possible mechanism by which combinatorial patterns of exon selection are achieved for alternatively spliced pre-mRNAs.

scholarly journals Reconstitution of the U1 small nuclear ribonucleoprotein particle.

1987 ◽  
Vol 7 (11) ◽  
pp. 4030-4037 ◽  
Author(s):  
J R Patton ◽  
R J Patterson ◽  
T Pederson
Keyword(s):  
Buoyant Density ◽  
Sedimentation Coefficient ◽  
Ribonucleoprotein Particle ◽  
Small Nuclear Ribonucleoprotein ◽  
In Vitro Reconstitution ◽  
U1 Snrnp ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoprotein Particle ◽  
Nuclease Sensitivity

Although the U1 small nuclear ribonucleoprotein particle (snRNP) was the first mRNA-splicing cofactor to be identified, the manner in which it functions in splicing is not precisely understood. Among the information required to understand how U1 snRNP participates in splicing, it will be necessary to know its structure. Here we describe the in vitro reconstitution of a particle that possesses the properties of native U1 snRNP. 32P-labeled U1 RNA was transcribed from an SP6 promoter-human U1 gene clone and incubated in a HeLa S100 fraction. A U1 particle formed which displayed the same sedimentation coefficient (approximately 10S) and buoyant density (1.40 g/cm3) as native U1 snRNP. The latter value reflects the ability to withstand isopycnic banding in Cs2SO4 without prior fixation, a property shared by native U1 snRNP. The reconstituted U1 particle reacted with both the Sm and RNP monoclonal antibodies, showing that these two classes of snRNP proteins were present. Moreover, the reconstituted U1 snRNP particle was found to display the characteristic Mg2+ switch of nuclease sensitivity previously described for native U1 snRNP: an open, nuclease-sensitive conformation at a low Mg2+ concentration (3 mM) and a more compact, nuclease-resistant organization at a higher concentration (15 mM). The majority of the U1 RNA in the reconstituted particle did not contain hypermethylated caps, pseudouridine, or ribose 2-O-methylation, showing that these enigmatic posttranscriptional modifications are not essential for reconstitution of the U1 snRNP particle. The extreme 3' end (18 nucleotides) of U1 RNA was required for reconstitution, but loop II (nucleotides 64 to 77) was not. Interestingly, the 5' end (15 nucleotides) of U1 RNA that recognizes pre-mRNA 5' splice sites was not required for U1 snRNP reconstruction.

scholarly journals U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor of 65 kDa, U2AF65, can promote U1 snRNP recruitment to 5′ splice sites

2003 ◽  
Vol 372 (1) ◽  
pp. 235-240 ◽  
Author(s):  
Patrik FÖRCH ◽  
Livia MERENDINO ◽  
Concepción MARTÍNEZ ◽  
Juan VALCÁRCEL
Keyword(s):  
Splice Sites ◽  
Ribonucleoprotein Particle ◽  
Rich Domain ◽  
Small Nuclear Ribonucleoprotein ◽  
U2 Snrnp ◽  
U1 Snrnp ◽  
Nuclear Ribonucleoprotein ◽  
Auxiliary Factor ◽  
Small Nuclear Ribonucleoprotein Particle

The splicing factor U2AF65, U2 small nuclear ribonucleoprotein particle (snRNP) auxillary factor of 65 kDa, binds to pyrimidine-rich sequences at 3′ splice sites to recruit U2 snRNP to pre-mRNAs. We report that U2AF65 can also promote the recruitment of U1 snRNP to weak 5′ splice sites that are followed by uridine-rich sequences. The arginine- and serine-rich domain of U2AF65 is critical for U1 recruitment, and we discuss the role of its RNA–RNA annealing activity in this novel function of U2AF65.

Reconstitution of the U1 small nuclear ribonucleoprotein particle

1987 ◽  
Vol 7 (11) ◽  
pp. 4030-4037
Author(s):  
J R Patton ◽  
R J Patterson ◽  
T Pederson
Keyword(s):  
Buoyant Density ◽  
Sedimentation Coefficient ◽  
Ribonucleoprotein Particle ◽  
Small Nuclear Ribonucleoprotein ◽  
In Vitro Reconstitution ◽  
U1 Snrnp ◽  
Nuclear Ribonucleoprotein ◽  
Small Nuclear Ribonucleoprotein Particle ◽  
Nuclease Sensitivity

Although the U1 small nuclear ribonucleoprotein particle (snRNP) was the first mRNA-splicing cofactor to be identified, the manner in which it functions in splicing is not precisely understood. Among the information required to understand how U1 snRNP participates in splicing, it will be necessary to know its structure. Here we describe the in vitro reconstitution of a particle that possesses the properties of native U1 snRNP. 32P-labeled U1 RNA was transcribed from an SP6 promoter-human U1 gene clone and incubated in a HeLa S100 fraction. A U1 particle formed which displayed the same sedimentation coefficient (approximately 10S) and buoyant density (1.40 g/cm3) as native U1 snRNP. The latter value reflects the ability to withstand isopycnic banding in Cs2SO4 without prior fixation, a property shared by native U1 snRNP. The reconstituted U1 particle reacted with both the Sm and RNP monoclonal antibodies, showing that these two classes of snRNP proteins were present. Moreover, the reconstituted U1 snRNP particle was found to display the characteristic Mg2+ switch of nuclease sensitivity previously described for native U1 snRNP: an open, nuclease-sensitive conformation at a low Mg2+ concentration (3 mM) and a more compact, nuclease-resistant organization at a higher concentration (15 mM). The majority of the U1 RNA in the reconstituted particle did not contain hypermethylated caps, pseudouridine, or ribose 2-O-methylation, showing that these enigmatic posttranscriptional modifications are not essential for reconstitution of the U1 snRNP particle. The extreme 3' end (18 nucleotides) of U1 RNA was required for reconstitution, but loop II (nucleotides 64 to 77) was not. Interestingly, the 5' end (15 nucleotides) of U1 RNA that recognizes pre-mRNA 5' splice sites was not required for U1 snRNP reconstruction.

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