L17 PT2 Splicing1

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Information about L17 PT2 Splicing1

Published on October 15, 2007

Author: Irvette

Source: authorstream.com

RNA Splicing:  RNA Splicing RNA splicing is the removal of intervening sequences (IVS) that interrupt the coding region of a gene Excision of the IVS (intron) is accompanied by the precise ligation of the coding regions (exons) Discovery of Split Genes (1977):  Discovery of Split Genes (1977) P. Sharp and R. Roberts - 1993 Nobel Prize in Physiology & Medicine Discovered using R-loop Analysis Cloned genomic DNAs of a few highly expressed nuclear genes (e.g., hemoglobin, ovalbumin), and certain Adenoviral genes were hybridized to RNA fractions and visualized by EM Loops form from RNA annealing to the template strand and displacing coding strand of DNA Slide3:  Genomic DNA fragment containing a Globin gene was annealed to large heterogenous nuclear RNA (hnRNA), which contained globin mRNA precursors. Fig. 14.3a Dotted line is RNA DNA coding Template strand Slide4:  When genomic globin gene was annealed to cytoplasmic mRNA (which contained mature globin mRNA) got an internal loop of single-stranded DNA (= spliced out intron). Fig. 14.3b template strand Coding strand Intron Classes & Distribution:  Intron Classes & Distribution Group I - common in organelles, nuclear rRNA genes of lower eukaryotes, a few prokaryotes Group II - common in organelles, also in some prokaryotes and archaea Nuclear mRNA (NmRNA) - ubiquitous in eucaryotes Nuclear tRNA- some eucaryotes Relationships of the 4 Intron Classes :  Relationships of the 4 Intron Classes Each class has a distinctive structure The chemistry of splicing of Groups I, II and NmRNA is similar – i.e, transesterification reactions The splicing pathway for Group II and nuclear mRNA introns are similar Splicing of Groups I, II and possibly NmRNA introns are RNA-catalyzed Self-Splicing Introns :  Self-Splicing Introns Some Group I and Group II introns can self- splice in vitro in the absence of proteins (or other RNAs), i.e. they are ribozymes. Each group has a distinctive, semi-conserved secondary structure. Both groups require Mg2+ to fold into a catalytically active ribozyme. Group I introns also require a guanosine nucleotide in the first step. Tetrahymena rRNA Group I Intron:  Tetrahymena rRNA Group I Intron First self-splicing intron; was discovered by T. Cech’s lab in 1981 In the 26S rRNA gene in Tetrahymena (a protist) First example of a catalytic RNA! Nobel Prize in Chemistry to T. Cech and S. Altman (showed that RNase P was a true “turnover” riboenzyme in vivo), 1989 Group I splicing mechanism:  Group I splicing mechanism GOH – guanosine nucleotide, guanosine will work because the phosphates don’t participate in the reaction. In vivo, GTP probably used. The 3’ terminal G of the intron is nearly 100% conserved. Cr.LSU intron: 2ndary structure of a group I intron:  Cr.LSU intron: 2ndary structure of a group I intron Old style drawing Newer representation Conserved core 5’ splice site Exon seq. in lower case and boxed Shows how splice sites can be brought close together by “internal guide sequence”. 3-D Model of Tetrahymena rRNA Intron:  3-D Model of Tetrahymena rRNA Intron Catalytic core consists of two stacked helices domains: 1. P5 – P4 – P6 –P6a (in green) 2. P9 – P7 – P3 – P8 (in purple) The “substrate is the P1 – P10 domain (in red and black), it contains both the 5’ and 3’ splice sites. Guanosine binding site of Group I Introns:  Guanosine binding site of Group I Introns It is mainly the G of a G-C pair in the P7 helix of the conserved core. It is highly specific for Guanosine (Km ~20 μM). Binds free GTP in the first splicing step. Binds the 3’-terminal G of the intron in the second splicing step. Splicing Factors for Self-Splicing Introns:  Splicing Factors for Self-Splicing Introns Some Group I and many Group II introns can’t self-splice in vitro (need protein factors?) Even self-splicing introns get help from proteins in vivo Based on fungal (yeast and Neurospora) mutants deficient in splicing of mitochondrial introns (respiratory-deficient) Protein splicing factors for Group I introns:  Protein splicing factors for Group I introns 2 types: Intron-encoded (promote splicing of only the intron that encodes it) Nuclear-encoded (for organellar introns) Nuclear-encoded ones function by: Promoting correct folding of the intron (CBP2 promotes folding of a cytochrome b intron) Stabilizing correctly folded structure (cyt18 promotes activity of a number of Group I introns) Cyt18 is also the mt tyrosyl-tRNA synthetase, dual-function protein Evolved from a tyrosyl-tRNA synthetase by acquiring a new RNA-binding surface Slide15:  Figure 5. Models of CYT-18/ΔC424-669 with Bound RNA Substrates(A) Dimeric CYT-18/ΔC424-669 with the T. thermophilus tRNATyr (orange) docked as in the T. thermophilus TyrRS/tRNATyr cocrystal structure (Yaremchuk et al. 2002; PDB ID: 1H3E). Subunits A (Sub. A; magenta) and B (Sub. B; blue) are defined as those that bind the tRNA acceptor and anticodon arms, respectively. Side chains at positions that did or did not give specific EPD-Fe-induced cleavages in the ND1 intron are shown in space-filling representations colored yellow and black, respectively.(B) Stereoview of dimeric CYT-18/ΔC424-669 with docked ND1 intron RNA. The model is based on optimized fit to directed hydroxyl radical cleavage data summarized in Figure 4B. The ND1 intron RNA (residues 27–182) is shown as a green ribbon, with purple balls indicating phosphate-backbone protections from full-length CYT-18 protein (Caprara et al., 1996a), and red ribbon segments indicating EPD-Fe cleavage sites. The C-terminal domain of T. thermophilus TyrRS (yellow) is shown positioned on subunit B as in the T. thermophilus/tRNATyr cocrystal structure (Yaremchuk et al., 2002). Slide17:  6 domains, “Helical Wheel” Domain I contains binding sites for the 5’ exon (keeps the 5’ exon from floating away after the first splicing step) Consensus Group II intron Structure Slide18:  Group II Splicing Pathway (1) The 2’ OH of a special internal A attacks the 5’ splice site creating a branched intron structure. (2) The 3’ OH of the 5’ exon attacks the 3’ splice site, ligating the exons and releasing the intron as a lariat structure. Structure of NmRNA Introns:  Structure of NmRNA Introns Most begin with GU and end with AG. Most of the internal sequences are not conserved. However, there are other important consensus sequences near the ends (in addition to GU and AG). Consensus Mammalian NmRNA Splice Signals:  Consensus Mammalian NmRNA Splice Signals 5’ ag/GUAAGU -------YNCURAC---YnNAG/g 3’ Y - pyrimidine (U or C) Yn - string of ~ 9 pyrimidines R - purine (A or G) N - any base Branch site sequence Nm RNA Splicing Mechanism :  Elucidating the overall mechanism & the cis elements and trans factors depended on: Site-directed mutagenesis of genes in vitro, and subsequent expression in vivo (yeast and Hela cells) Development of accurate splicing extracts (HeLa cells and yeast) Isolation of temperature-sensitive yeast mutants defective in NmRNA splicing Nm RNA Splicing Mechanism Slide22:  Figs. 14.5 & 6 Kinetics of In vitro Splicing in a Hela cell nuclear extract Pre - radioactively labeled precursor RNA The splicing reactions were separated by gel electrophoresis. Notice that the intron and intron-exon RNAs have an unusually reduced mobility in these polyacrylamide-urea gels. Slide23:  Fig. 14.6b Plot of changes in amounts of products and intermediates during the splicing reaction in the previous slide. Slide24:  2 Transesterification reactions w/phosphates 1st - 2’OH of internal A attacks the phosphodiester that links the 5’ end of the intron to exon 1, producing lariat and 5’ exon molecules 2nd - 3’OH of the 5’ exon attacks the phosphodiester that links the beginning of exon 2 and the 3’ end of th intron, ligating the exons Intron released as lariat - No. of phosphodiesters is conserved! In yeast, the branch-point sequence determines which downstream AG is used.:  In yeast, the branch-point sequence determines which downstream AG is used. Branch sequence Exon 1 Exon 2 branch Fig. 14.9 RNAs that were tested for splicing in vivo. Inserted sequence Branch site moved into exon 2.

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