UBAIII Biologia Molecular

Apresentação em tema: "UBAIII Biologia Molecular"— Transcrição da apresentação:

1 UBAIII Biologia Molecular
1º Ano 2013/2014

2 Sumário: Capítulo V. Síntese e processamento do RNA mensageiro.
O processamento do mRNA eucariota Implicações evolutivas dos intrões e splicing Capítulo VI. RNAs não codificantes e de interferência Micro RNAs e a descoberta de novas formas de regulação: iRNA miRNA piwiRNA MJC-T06 31/out/2013

3 Exões e intrões MJC-T06 31/out/2013

4 Evidência experimental de splicing
cDNA Enzimas de restrição MJC-T06 31/out/2013

5 Processamento do pré-mRNA
Splicing MJC-T06 31/out/2013

6 Processamento do mRNA MJC-T06 31/out/2013

7 Intrões do grupo II – Self-splicing
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8 Splicing do grupo II MJC-T06 31/out/2013

9 Spliceossoma – Splicing mediado por snRPs
Animação Figure 5.32 Schematic model of the assembly of the splicing machinery and some of the steps that occur during pre-mRNA splicing. Step 1 shows the portion of the pre-mRNA to be spliced. In step 2, the first of the splicing components, U1 snRNP, has become attached at the 5’ splice site of the intron. The nucleotide sequence of U1 snRNA is complementary to the 5’ splice site of the pre-mRNA, and evidence indicates that U1 snRNP initially binds to the 5’ side of the intron by the formation of specific base pairs between the splice site and U1 snRNA (see inset A). The U2 snRNP is next to enter the splicing complex, binding to the pre-mRNA (as shown in inset A) in a way that causes a specific adenosine residue (dot) to bulge out of the surrounding helix (step 3). This is the site that later becomes the branch point of the lariat. U2 is thought to be recruited by the protein U2AF, which binds to the polypyrimidine tract near the 3’ splice site. U2AF also interacts with SR proteins that bind to the exonic splicing enhancers (ESEs). These interactions play an important role in recognizing intron/exon borders. The next step is the binding of the U4/U6 and U5 snRNPs to the premRNA with the accompanying displacement of U1 (step 4). The assembly of a spliceosome involves a series of dynamic interactions between the pre-mRNA and specific snRNAs and among the snRNAs themselves. As they enter the complex with the pre-mRNA, the U4 and U6 snRNAs are extensively base-paired to one another (inset B). The U4 snRNA is subsequently stripped away from the duplex, and the regions of U6 that were paired with U4 become base-paired to a portion of the U2 snRNA (inset C). Another portion of the U6 snRNA is situated at the 5’ splice site (inset C), having displaced the U1 snRNA that was previously bound there (inset A). It is proposed that U6 is a ribozyme and that U4 is an inhibitor of its catalytic activity. According to this hypothesis, once the U1 and U4 snRNA have been displaced, the U6 snRNA is in position to catalyze the two chemical reactions required for intron removal. According to an alternate view, the reactions are catalyzed by the combined activity of U6 snRNA and a protein of the U5 snRNP. Regardless of the mechanism, the first reaction (indicated by the arrow in inset C) results in the cleavage of the 5’ splice site, forming a free 5’ exon and a lariat intron–3’ exon intermediate (step 5). The free 5’ exon is thought to be held in place by its association with the U5 snRNA of the spliceosome, which also interacts with the 3’ exon (step 5). The first cleavage reaction at the 5’ splice site is followed by a second cleavage reaction at the 3’ splice site (arrow, step 5), which excises the lariat intron and simultaneously joins the ends of the two neighboring exons (step 6). Following splicing, the snRNPs must be released from the pre-mRNA, the original associations between snRNAs must be restored, and the snRNPs must be reassembled at the sites of other introns. MJC-T06 31/out/2013

10 Sequências indicadoras do local de splicing
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11 Estrutura de uma snRNP MJC-T06 31/out/2013
The structure of an snRNP. (A) Model of a U1 snRNP particle based on biochemical data and structural information obtained by cryoelectron microscopy. At the core of the particle is a ring-shaped protein complex composed of the seven different Sm proteins that are common to all U snRNPs. Three other proteins are unique to the U1 snRNP (named 70K, U1-A, and U1-C). Stems I, II and IV are parts of the 165-base U1 snRNA. The snRNP is assembled in the cytoplasm and imported into the nucleus, where it carries out its function. (B) A model of the U1 snRNA in the same orientation as in A. MJC-T06 31/out/2013

12 Síntese e Processamento de mRNA
Figure 5.34 Schematic representation of a mechanism for the coordination of transcription, capping, polyadenylation, and splicing. In this simplified model, the C-terminal domain (CTD) of the large subunit of the RNA polymerase serves as a flexible scaffold for the organization of factors involved in processing pre-mRNAs, including those for capping, polyadenylation, and intron removal. In addition to the proteins depicted here, the polymerase is probably associated with a host of transcription factors, as well as enzymes that modify the chromatin template. The proteins bound to the polymerase at any particular time may depend on which of the serine residues of the CTD are phosphorylated. The pattern of phosphorylated serine residues changes as the polymerase proceeds from the beginning to the end of the gene being transcribed. The phosphate groups linked to the #5 residues are largely lost by the time the polymerase has transcribed the 3’ end of the RNA. © 2013 John Wiley & Sons, Inc. All rights reserved.

13 Processing the ovomucoid pre-mRNA as determined by northern blot
Splicing de RNA O RNA é uma molécula que pode assumer as funções estruturais e de catálise. As funções de catálise são assumidas pelas riboenzimas. Estas reações pelas quais se cortam e colam mRNAs pode o denominado “Exon shuffling” dá origem a novidade genética Figure 5.35 Processing the ovomucoid pre-mRNA. The photograph shows a technique called a Northern blot in which extracted RNA (in this case from the nuclei of hen oviduct cells) is fractionated by gel electrophoresis and blotted onto a membrane filter. The immobilized RNA on the filter is then incubated with a radioactively labeled cDNA (in this case a cDNA made from the ovomucoid mRNA) to produce bands that reveal the position of RNAs containing the complementary sequence. The mature mRNA encoding the ovomucoid protein is 1100 nucleotides long and is shown at the bottom of the blot. It is evident that the nucleus contains a number of larger-sized RNAs that also contain the nucleotide sequence of the ovomucoid mRNA. The largest RNA on the blot has a length of 5450 nucleotides, which corresponds to the size of the ovomucoid transcription unit; this RNA is presumably the primary transcript from which the mRNA is ultimately carved. Other prominent bands contain RNAs with lengths of 3100 nucleotides (which corresponds to a transcript lacking introns 5 and 6), 2300 nucleotides (a transcript lacking introns 4, 5, 6, and 7), and 1700 nucleotides (a transcript lacking all introns except number 3). Processing the ovomucoid pre-mRNA as determined by northern blot © 2013 John Wiley & Sons, Inc. All rights reserved.

14 Bibliografia Capítulo 5 secção 5.4 e 5.5 Capítulo 30 Lippincott’s
MJC-T06 31/out/2013