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Post-transcriptional modifications: splicing Eukaryotic cells modify their RNA after transcription RNA transcripts in eukaryotes are modified, or processed, before leaving the nucleus to yield functional mRNA. Eukaryotic RNA transcripts can be processed in two ways: 1. Covalent alteration of both the 3’ and 5’ ends. 2. Removal of intervening sequences. Primary transcript – general term for initial RNA transcribed from DNA Pre-mRNA – primary transcript that will be processed to functional mRNA 1 Eukaryotic cells modify their RNA after transcription RNA processing: RNA splicing 2 Split genes and RNA splicing RNA splicing – RNA processing that removes introns and joins exons from eukaryotic pre-mRNA; produces mature mRNA that will move into the cytoplasm from the nucleus. •Enzymes excise introns and splice exons to form mRNA with continuous coding sequence. •RNA splicing also occurs during post-transcriptional processing of tRNA and rRNA. RNA splicing Spliceosome – a large molecular complex that catalyzes RNA splicing reactions. •As the spliceosome is assembled, one type of snRNP base pairs with a complementary sequence at the 5’ end of the intron. •The spliceosome precisely cuts the RNA transcript at the specific splice sites at either end of the intron, which is excised as a loop. •The intron is released and the adjacent exons are immediately spliced together by a spliceosome. 3 RNA splicing Each end of the intron has short boundary sequences that accurately signal the RNA splicing sites. Small nuclear ribonucleoproteins (snRNPs) – complexes of proteins and small nuclear RNAs that are found only in the nucleus. Some participate in RNA splicing. They are composed of: Small nuclear RNA – this molecule has less than 300 nucleotides. Protein – each snRNP possesses several different proteins. There are different types of snRNPs with different functions. The roles of snRNPs (small nuclear ribonuclearproteins) and spliceosomes in mRNA splicing. 4 The R-looping experiments reveal the introns of adevovirus. Loops-introns of the gene that can not hybridize to DNA Most of higher eukaryotic genes coding for mRNA, tRNA and some coding for rRNA are interrupted by unrelated regions called introns. The other parts of the genes are called exons. Exons contain information that appears in the functional mRNA Genes for mRNA have 0 to 60 introns Genes for tRNA have 0 to 1 intron. 5 mRNA synthesis 1 – primary transcript, hnRNA; contains introns 2 – mRNA maturation – part of maturation – removal of introns, splicing How does the cell deal with introns during transcription? The process of cutting out the introns from immature RNA and stitching together the exons is called splicing The outline of splicing 6 The introns are transcribed. Experiment with mouse beta-globin mRNA and its precursor. precursor mRNA Splicing signals are important •Splicing has to be precise. •Splicing signals in nuclear mRNA are remarkably uniform •The first two bases of intron are always GU. •The last two are always AG. •The 5’ and 3’ splice sites have consensus sequences that extend beyond the GU and AG motifs. •The consensus sequences are important for splicing •Mutations leads to abnormal splicing 7 Intron-exon boundaries Consensus sequence: 5’-AG/GUAAGU-intron-YNCURAC-YnNAG/G-3’ Y is pyrimidine C or U Yn – string of 9 pyrimidines R – purine, A or G A – special A that participates in forming branched splicing intermediate N – any base Yeast mRNA – a bit different from mammals 5’-/GUAUGU-intron-UACUAAC-YAG/-3’ Simplified mechanism of nuclear mRNA precursor 1. The 2’-hydroxyl group of adenine within the intron attacks the PDF bond linking the first exon to the intron. This attack breaks the bond between the exon 1 and intron, yielding the free exon 1 and the lariat-shaped intron-exon 2 intermediate with the GU at the 5’ end of the intron linked through the PDB to the breakpoint A. The lariat is a consequence of the internal attack of one part of the RNA precursor on another part of the same molecule. 8 Simplified mechanism of nuclear mRNA precursor 2. The free 3’-hydroxyl group on exon 1 attacks the PDF bond between the intron and exon2. This yields the spliced product and the lariat-shaped intron. NB – several lines of evidence demonstrate that nuclear mRNA precursors are spliced via lariat-shaped, or branched intermediate. A signal at the branch Special regions within introns are also important for splicing Experimental evidence: removal of a region between 35 and 70 bp upstream of the introns 3’ splice site blocked splicing Special intron regions contain the branch-point adenine nucleotide UACUAAC – in yeast Higher eukaryotes – sequence more variable. It appears to tell splicing machinery which AG to select as a 3’ splice site. 9 Splicing takes place on a particle called spliceosome Yeast and mammalian once have sedimentation coefficients 40S and 60S. Snurps – agents to recognize the critical splicing signals. snRNPs – U1,U2, U4, U5 and U6. Splicing substrate+U1= CC U2, ATP A U4/U6 and U5 B1 U4 diss. From U6 U6 displ. U1 at 5’ splice site U1 and U4 exit U6 bps withU2 =B2 1st splicing stepC1 2nd splicing stepC2 Splicing RNA exits, Lariat held by I. 10 Alternative splicing is important Different RNA and thus protein can be produced: difference between membrane-bound and secreted protein Drosophila – of products 3 genes involved in sex determination are alternatively spliced Ribozymes Other kinds of RNA primary transcripts, such as some of those giving rise to tRNA and rRNA, are spliced by mechanisms that do not involve spliceosomes, however, as with mRNA splicing, RNA is often involved in catalyzing the reactions. Ribozymes – RNA molecules that can catalyze reactions by breaking and forming covalent bonds, they are called ribozymes to emphasize their catalytic activity. 11 RNA has catalytic capacities In early 1970 it was discovered that folded domains of RNA molecules have catalytic capacities just as the folds (helices and beta-strands) of proteins do. Less is understood about the 3D structure of the catalytic sites, but catalytic activities were identified. RNA has catalytic capacities Example - the ability of RNA to catalyze the cutting of an RNA chain. The example of the mode of action of such ribozyme (RNA enzyme): the substrate chain is base-paired with a site on the ribozyme, and the cleavage is induced at the precise nucleotide in the substrate. Other reactions –eg. phosphotransferase reaction, which unites two distant segments of RNA chain after cleavage has occurred is called splicing. 5’ G C G C C 3’ C G C G G A G C A C A AA A G U G U G U U AG Nicking site C Substrate U C G A G C 3’ C A G C U C G 5’ Ribozyme 12 Functional and evolutionary importance of introns Introns may play important role in the cells. Intron sequences direct the synthesis of different proteins and may control gene activity. The splicing process itself may help to regulate the export of mRNA to the cytoplasm. Introns may allow single gene to direct the synthesis of different proteins. This can occur if the same transcript is processed differently among various cell types in the same organism. Functional and evolutionary importance of introns All introns may be removed from particular transcript in one case, but in the other one or more introns may be left in place. The proteins will be different. Introns play important roles in the evolution of protein diversity they increase the probability that recombination of exons will occur between alleles. 13 Functional and evolutionary importance of introns In split genes, coding sequences can be separated by long distances, so they have higher recombination frequencies than continuously coded genes without introns. Exons of the split gene may code for different domains of a protein that have specific functions, such as enzyme’s active site or a protein’s binding site. Protein domains – continuous polypeptide sequences that are structural and functional units in proteins with a modular architecture. Alteration of pre-mRNA ends During pre-mRNA processing, both the 5’ and 3’ ends are covalently modified. 5’ cap – Modified guanine nucleotide (guanosine triphosphate) that is added to the 5’ end of mRNA shortly after transcription begins. It has two important functions: •Protects the growing mRNA from degradation by hydrolytic enzymes. •Help small ribosomal subunits recognize the attachment site on mRNA’s 5’ ends. A leader segment of mRNA may also be part of the ribosome recognition signal. Leader signal – Non-coding (untranslated) sequence of mRNA from the 5’ end to the start codon. 14 Alteration of pre-mRNA ends The 3’ end, which is transcribed the last, is modified by enzymatic addition of a poly-A-tail, before the mRNA exits the nucleus. Poly-A tail – sequence of about 0 to 200 adenine nucleotides added to the 3’ end of mRNA before it exits the nucleus. •May inhibit degradation of mRNA in the cytoplasm •May facilitate attachment to the small ribosomal subunit •May regulate protein synthesis by facilitating mRNA’s export from the nucleus to the cytoplasm •Is not attached directly to the stop codon, but to untranslated trailer segment of mRNA. Trailer sequence – noncoding (untranslated) sequence Reading: Pages 425-465 15