A series of efficiently spliced pre-mRNA substrates containing single 4-thiouridine residues were used to monitor RNA-protein interactions involving the branch site-3 splice site-3 exon region during yeast premRNA splicing through cross-linking analysis. Prior to the assembly of the prespliceosome, Mud2p and the branch point bridging protein cross-link to a portion of this region in an ATP-independent fashion. Assembly of the prespliceosome leads to extensive cross-linking of the U2-associated protein Hsh155p to this region. Following the first step of splicing and in a manner independent of Prp16p, the U5 small nuclear ribonucleoprotein particle-associated protein Prp8p also associates extensively with the branch site-3 splice site-3 exon region. The subsequent cross-linking of Prp16p to the lariat intermediate is restricted to the 3 splice site and the adjacent 3 exon sequence. Using modified substrates to either mutationally or chemically block the second step, we found that the association of Prp22p with the lariat intermediate represents an authentic transient intermediate and appears to be restricted to the last eight intron nucleotides. Completion of the second step leads to the cross-linking of an unidentified ϳ80-kDa protein near the branch site sequence, suggesting a potential role for this protein in a later step in intron metabolism. Taken together, these data provide a detailed portrayal of the dynamic associations of proteins with the branch site-3 splice site region during spliceosome assembly and catalysis.The locations of introns within nascent yeast pre-mRNAs is specified largely by just three short conserved sequences located at and near the 5Ј and 3Ј ends of the intron (6, 47). These sequences are recognized during the assembly of the spliceosome, a massive macromolecular complex consisting of the U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs) and at least 60 associated proteins (31, 57). The spliceosome not only must precisely recognize intron boundary sequences to avoid the introduction of catastrophic frameshift errors in coding regions but also must be able to accommodate significant variations in intron size, sequence, and organization. A full understanding of how accurate splice site recognition is achieved will require detailed knowledge of the dynamic contacts between the conserved sequences that define intron boundaries and the components of the spliceosome.Nuclear pre-mRNA splicing proceeds via two sequential transesterification reactions. In the first catalytic step, the 2Ј hydroxyl group of the last adenosine residue in the conserved branch site sequence (UACUAAC) attacks the phosphodiester bond at the 5Ј splice site to yield free 5Ј exon and branched lariat intermediates. The branch site and 5Ј splice site sequences are initially recognized by binding of the branch point bridging protein (BBP) (5) and the U1 snRNA (45), respectively. These sequences are then recognized a second time through interactions involving the U2 and U6 snRNAs prior to the first c...
The extracellular presence of endotoxinfree heat shock protein 70 (HSP70) enhances the rate and capacity of macrophage-mediated phagocytosis at 6 times the basal rate. It is protein-specific, doseand time-dependent and involves the internalization of inert microspheres, Grampositive and -negative bacteria and fungi. Structurally, exogenous HSP70 binds the macrophage plasma membrane, specifically on its lipid raft-microdomain. Disruption of lipid rafts, HSP70-LR interaction, or denaturing HSP70 abrogates the HSPmediated increase in phagocytosis. Further, HSP70-mediated phagocytosis directly enhances the processing and presentation of internalized antigens via the endocytic MHC class-II pathway to CD4 ؉ T lymphocytes. Modulating the HSP70-LR interaction presents an opportunity to intervene at the level of hostpathogen interface: a therapeutic tool for emerging infections, especially when conventional treatment with antibiotics is ineffective (antibiotic resistance) or unavailable (rapidly spreading, endemic). These results identify a new role for HSP70, a highly conserved molecule in stimulating phagocytosis: a primordial macrophage function, thereby influencing both innate and adaptive immune responses. (Blood.
Using site-specific incorporation of the photo-chemical cross-linking reagent 4-thiouridine, we demonstrate the previously unknown association of two proteins with yeast 3' splice sites. One of these is an unidentified approximately 122 kDa protein that cross-links to 3' splice sites during formation of the pre--spliceosome. The other factor is the DExH-box RNA helicase, Prp22p. With substrates functional in the second step of splicing, only very weak cross-linking of Prp22p to intron sequences at the 3' splice site is observed. In contrast, substrates blocked at the second step exhibit strong cross-linking of Prp22 to intron sequences at the 3' splice site, but not to adjacent exon sequences. In vitro reconstitution experiments also show that the association of Prp22p with intron sequences at the 3' splice site is dependent on Prp16p and does not persist when release of mature mRNA from the spliceosome is blocked. Taken together, these results suggest that the 3' splice site of yeast introns is contacted much earlier than previously envisioned by a protein of approximately 120 kDa, and that a transient association of Prp22p with the 3' splice site occurs between the first and second catalytic steps.
mice. These results demonstrate that C/EBP deletion decreases plasma FFA levels and increases insulin signal transduction specifically in skeletal muscle, and both contribute to increased whole-body insulin sensitivity.
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