Interrupted Genes Prokaryotes – continuous gene(uninterrupted) Eukaryotes...

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Transcript of Interrupted Genes Prokaryotes – continuous gene(uninterrupted) Eukaryotes...

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RNA SPLICINGSUBMITTED BY SELMA ABDUL SAMAD BCH-10-05-02 S3 MSc BIOCHEMISTRYInterrupted GenesProkaryotes continuous gene(uninterrupted) Eukaryotes gene is interrupted with non-coding sequences (introns)RNA splicing the removal of these introns while joining the restTerminologyExons sequences represented in mature RNA(A gene starts and ends with exons that correspond to the 5 and 3 ends of RNA)Introns Intervening sequences that are removed when the primary transcript is processed to the mature RNADNA TRANSCRIPT(RNA COPY) MATURE RNA

The mechanism excludes any splicing together of sequences representing different alleles A typical mammalian gene has 7 to 8 exons spread out over ~16 kb . The exons are relatively short(~100 200 bp) and introns relatively long(>1 kb)So, gene is interrupted while mRNA(~2.2 kb) is uninterrupted , which requires the primary transcript(pre-mRNA) to be processed.Nuclear RNA(including pre-mRNA) - much larger than mRNA - very unstable - much greater sequence complexity - known as hnRNA(heterogenous nuclear RNA)

hnRNP ribonuclear protein ; the physical form of hnRNA,which is bound to proteins ; Has the form of beads connected by a fiber.Splicing and other post transcriptional modifications take place in the nucleus

SPLICING IS OF SEVERAL TYPESIn higher eukaryotes introns removed by a system that recognizes only short consensus sequences conserved at exon-intron boundaries and within the intron. - Requires spliceosomes (large splicing apparatus) - Mechanism involves transesterifications - Catalytic center includes RNA and proteins.Autonomous splicing of introns by certain RNAs - 2 types of introns distinguished by 20 and 30 structures - Mechanism transesterification - Catalytic agent RNA (catalytic RNA)Splicing of yeast tRNA accomplished by enzymes that use cleavage and ligation.

SPLICE JUNCTIONS (splice sites)The two exon-intron boundaries that include the sites of breakage and reunion.ie, the junction between exons and introns.There is no extensive homology or complementarity between 2 ends of an intron. But there are well conserved,short,consensus sequences.High conservation is found only immediately within the intron at the presumed junctions.

GT-AG rule ie., GU-AG rule in pre-mRNA - An intron starts with dinucleotide GU and ends in AG - called 5 and 3 splice sites resp.

SPLICE JUNCTIONS ARE READ IN PAIRS In an mRNA, introns are multiple and longAppropriate 5 and 3 sites should be paired - It could be an intrinsic property of RNA to connect the sites at the ends of a particular intron - Splicing could follow rules that ensure a 5 site is always paired to a following 3 site

In principle any 5 splice site can be connected to any 3 splice site. So,there are preferred pathways that ensure right splicing.The conformation of the RNA influences the accessibility of the splice sites. As particular introns are removed,the conformation changes and new pairs of splice sites become available.So , the splicing reaction does not proceed sequentially along the precursor RNA.

SPLICING PROCEEDS THROUGH A LARIATSplicing is independent of transcription or other post transcriptional modifications,yet occur co-ordinated.In vivo, the exons are not released as free molecules during splicing,but remain held together by the splicing apparatus.Splicing requires the 5 and 3 splice sites and a branch site just upstream of the 3 splice site.Steps in splicing - A cut is made at the 5 splice site, separating the left exon and the right intron-exon molecule SPLICING PROCEEDS THROUGH A LARIAT

- The left exon becomes linear- The right intron-exon molecule form a lariat by forming a 5-2 bond between 5 terminus and the target base A called the branch site - The 3 splice site is then cut releasing free intron in the lariat form - The right exon is ligated (spliced) to the left exon - The lariat is then debranched to give a linear excised intron which is rapidly degradedThe branch site plays an important role in identifying the 3 splice site. The consensus is highly conserved in yeast as UACUAAC.The branch site is not well conserved in higher eukaryotes, but has a preference for bases at each position and retains the target A nucleotide.The branch site lies 18 to 40 nucleotides upstream of the 3 splice site.The lariat formation is effected by transesterification - First, a nucleophilic attack by the 2-OH of the invariant A on the 5 splice site - Second, the free 3-OH of the exon that was released , now attacks the bond at the 3 splice siteTHE SPLICING APPARATUSContains both proteins and RNAs ; Splicing occurs only after all components are sequentially assembled on the pre-mRNAThe small RNAs are found both in nucleus and cytoplasm of eukaryotic cellsIn nucleus small nuclear RNAs (snRNAs)In cytoplasm small cytoplasmic RNAs (scRNAs)In nucleolus snoRNAs They exist as ribonucleoprotein particles snRNPs and scRNPs (known colloq. as snurps and scyrps)Spliceosome large particulate complex formed of snRNPs involved in splicing and many additional proteins - It comprises a 50S to 60S RNP particle

Like the ribosome, the spliceosome depends on RNA-RNA, protein-RNA and protein-protein interactions.The 5 snRNPs involved in splicing are U1, U2, U5, U4 and U6 . Each snRNP contains a single snRNA and several(>20) proteins. U4 and U6 are usually found as a single U4/U6 particle.

SPLICEOSOME MACHINERYBefore any irreversible change is made to the RNA, all of the splicing components are assembled and have ensured that the splice sites are available.Splicing is divided into 2 stagesa) The 5 splice site, branch sequence and adjacent pyrimidine tract are recognised.The spliceosome complex is assembledb) Structure of transcript is changed by cleavage and ligation. Components of the complex are released or reorganised as it proceeds through the reactions.Binding of U1 snRNP to the 5 splice site is the first step in splicing. ie.,one of its proteins,U1-70k interacts with protein ASF/SF2(an SR class general splicing factor) causing U1 snRNA to base pair with the 5 site by a single stranded region at 5 terminus (4 to 6 bases complementary with splice site).Complementarity between U1 snRNA and 5 splice site is necessary for splicing, with pairing stabilized by proteins of U1 snRNP. [SR proteins imp. group of splicing factors & regulators - Take their name from Ser-Arg rich region with variable length. They interact each other via these regions. They bind RNA/connects U2AF to U1. They are essential part of spliceosome,forming a framework on RNA substrate]

The first complex formed during splicing is the E (early presplicing) complex it contains U1 snRNP,U2AF(a splicing factor) and some SR proteins.The formation of E complex identifies a pre-mRNA as a substrate for formation of splicing complex and is hence also called the commitment complex.In the E complex, U2AF is bound to the region between the branch site and the 3' splice site. In most organisms, it has a large subunit (U2AF65) that contacts a pyrimidine tract downstream of the branch site; a small subunit (V2AF35) directly contacts the dinucleotide AG at the 3' splice site.

Another splicing factor, called SF1in mammals and BBP in yeast. connects V2AF/Mud2 to the U1 snRNP bound at the 5' splice site. Complex formation is enhanced by the cooperative reactions of the two proteins; SF 1 and U2AF (or BBP and Mud2) bind together to the RNA substrate -1 Ox more effectively than either alone. This interaction is probably responsible for making the first connection between the two splice sites across the intron.The E complex is converted to the A complex when U2 snRNP binds to the branch site. Both UI snRNP and U2AF/Mud2 are needed for U2 binding. The U2 snRNA includes sequences complementary to the branch site.A sequence near the 5 end of the snRNA base pairs with the branch sequence in the intron. Several proteins of the U2 snRNP are bound to the substrate RNA just upstream of the branch site.The binding of U2 snRNP requires ATP hydrolysis and commits a pre-mRNA to the splicing pathway by generating A presplicing complex.

Formation of E complexIntron definition The two splice sites are recognised without requiring any sequences outside of the intron.The SR proteins may enable U2AFlU2 snRNP to bind in vitro in the absence of UI, raising the possibility that there could be a U1-independent pathway for splicingExon definition When introns are long and splice sites are weak ; sequences downstream of the intron itself are required ; The 3 splice site is recognized as part of a complex that forms across the next exon. though, in which the next 5' splice site is also bound by UI snRNA. This UI snRNA is connected by SR proteins to the U2AF at the pyrimidine tract.5 snRNPs Form the SpliceosomeThe snRNPs and factors associate with E complex in a defined order.B1 complex formed when a trimer U5 and U4/U6 binds to A complex(U1 and U2 snRNPs)This is the spliceosome complex has all components needed for splicing.B2 complex formed when U1 snRNA is released,other components,esp U6 comes into juxtaposition with 5 splice site, and U5 shifts to the vicinity of intron sequences.

The role of U4 snRNA may be to sequester U6 snRNA until it is needed. So U4 is released with hydrolysis of ATP ,triggering catalytic reaction.When U4 is released,the region of U6 initially base paired with U4 now is free. The first part of it pairs with U2; the second part forms an intramolecular hairpin.Thus several pairing reactions between snRNAs and the substrate RNA occur in the course of splicing.U6 snRNA is not used up in a splicing reaction and at completion must be released from U2 so tha