Showing papers by "Phillip A. Sharp published in 1994"

Branch nucleophile selection in pre-mRNA splicing: evidence for the bulged duplex model

Abstract: Selection of the nucleophile for the first step of nuclear pre-mRNA splicing was probed by site-specific incorporation into splicing substrates of nucleotides modified at the 2' position. The differing abilities of ribose, 2'-deoxyribose, and arabinose nucleotides to base-pair within an RNA.RNA duplex and to contribute a nucleophilic 2'-OH group were exploited to analyze the paired/unpaired disposition of the branch site nucleotide. The results provide direct evidence for a bulged duplex model in which either of two adjacent purines within the consensus branch site sequence may shift into a bulged position and contribute the 2'-OH group for the first step of splicing. Furthermore, the presence of a consensus branch site that cannot present a reactive nucleophile suppresses splicing, including the use of cryptic branch sites elsewhere. We conclude that the branch site region base-pairing with U2 snRNA determines the first step nucleophile and persists at the time of the first transesterification reaction.

Association of nuclear matrix antigens with exon-containing splicing complexes.

Abstract: mAbs raised against the human nuclear matrix (anti-NM)1 mAbs have been used to investigate the role of nuclear matrix antigens in pre-mRNA processing. The three anti-NM mAbs used in this study recognize antigens that are highly localized to nuclear matrix speckles. Surprisingly, all three of these mAbs preferentially immunoprecipitate splicing complexes containing exon sequences. The anti-NM mAbs efficiently immunoprecipitate the exon product complex but not complexes containing the lariat product after the second step of splicing. Two of the anti-NM mAbs completely inhibit pre-mRNA splicing in vitro. However, none of the anti-NM mAbs appear to recognize factors stably associated with splicing snRNPs. The three anti-NM mAbs predominantly react with distinct high molecular weight antigens, which belong to a class of nuclear proteins that selectively precipitate with Ser-Arg protein-splicing factors in the presence of high Mg2+ concentrations. Immunological, biochemical, and cell biological data indicate that two of the NM antigens are related to the defined set of Ser-Arg proteins. The results suggest the existence of an extended Ser-Arg family as a component of the nuclear matrix.

Complementation by SR proteins of pre-mRNA splicing reactions depleted of U1 snRNP.

Abstract: Individual small nuclear ribonucleoproteins (snRNPs) U1, U2, and U4/U6 were removed from nuclear extracts of HeLa cells by antisense affinity depletion. Addition of a highly purified preparation of SR proteins fully restored splicing activity in reactions depleted of U1 snRNP but did not reconstitute splicing in reactions depleted of the other snRNPs. Affinity selection experiments revealed that spliceosomes lacking U1 snRNA formed in the U1 snRNP-depleted reactions reconstituted with SR proteins. Thus, high concentrations of SR proteins facilitate the assembly of precursor messenger RNA (pre-mRNA) into a spliceosome in the absence of interactions with U1 snRNP.

Dynamic association of proteins with the pre-mRNA branch region.

Abstract: The association of proteins with the branch site region during pre-mRNA splicing was probed using a novel methodology to site-specifically modify the pre-mRNA with the photo-reagent benzophenone. Three sets of proteins were distinguished by the kinetics of their associations with pre-mRNAs, by their association with discrete splicing complexes, and by their differing factor requirements. An early U1 snRNP-dependent cross-link of the branch region to a p80 species was followed by cross-links to p14, p35, and p150 polypeptides associated with the U2 snRNP-pre-mRNA complex. Concomitant with formation of the spliceosome, a rearrangement of protein factors about the branch region occurred, in which the p35 and p150 cross-links were replaced by p220 and p70 species. These results establish that the branch region is recognized in a dynamic fashion by multiple distinct proteins during the course of spliceosomal assembly.

Homeodomain Determinants of Major Groove Recognition

Abstract: The homeodomain is a highly conserved structural module that binds DNA and participates in protein-protein interactions. Most homeodomains contain residues at positions 47 and 51 which mediate recognition of a TAAT core binding sequence in the major groove. The constraints imposed on the identity of these residues by homeodomain structure and DNA docking have been examined in the context of the POU domain of the Oct-1 transcription factor. A bacterial library, in which POU homeodomain residues 47 and 51 have been randomized, was probed on nitrocellulose filters for the binding of DNA fragments containing the consensus octamer sequence. The residues which provide for the highest affinity interaction with the octamer consensus sequence, and the greatest specificity, are the highly conserved wild-type residues valine 47 and asparagine 51. Interestingly, a class of variants containing arginine at position 51 was also detected in the screen and found to have moderate affinity for the consensus sequence but reduced specificity compared to the wild-type protein. A single variant containing arginine at both positions 47 and 51 was detected when the library was probed with fragments containing nucleotide substitutions at positions expected to be contacted by residues 47 and 51. This variant was used to alter the DNA-binding specificity of a transcriptional regulatory complex which depends upon Oct-1 for DNA recognition. These findings suggest that homeodomain structure and DNA docking constrain in the versatility of the domain in that only a limited set of amino acid determinants can endow the domain with specific, high-affinity DNA binding.