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![The hnRNA runs 5' 3' from the top left to lower right,
where transcription terminates
Upon recognition of the AAUAAA signal, the 3' end is
removed by an enzyme complex [CFI, II] at the cut site.
Another enzyme complex [PA] synthesizes a poly(A)
tail and ligates it to the free 3' of the pre-message
Post-transcriptional processing of mRNA; polyadenylation of 3' tail](https://image.slidesharecdn.com/posttranscriptionalmodifications-210708162934/75/Post-transcriptional-modifications-8-2048.jpg)
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Post transcriptional modifications
- 1. Post-transcriptional modifications of mRNA PrasannaR Kovath Assistant Professor Department of Biotechnology
- 2. Post-transcriptional modification orco-transcriptional modification is a set of biological processes common to most eukaryotic cells by which an RNA primary transcript is chemically altered following transcription from a gene to produce a mature, functional RNA molecule that can then leave the nucleus and perform any of a variety of different functions in the cell.
- 3. RNA Capping Isthe First Modification of Eukaryotic Pre-mRNAs • As soon as RNA polymerase II has produced about 25 nucleotides of RNA, the 5′ end of the new RNA molecule is modified by addition of a “cap” that consists of a modified guanine nucleotide • The capping reaction is performed by three enzymes acting in succession: • one (a phosphatase) removes one phosphate from the 5′ end of the nascent RNA, another (a guanyl transferase) adds a GMP in a reverse linkage (5′ to 5′ instead of 5′ to 3′), and a third (a methyl transferase) adds a methyl group to the guanosine. • Because all three enzymes bind to the phosphorylated RNA polymerase tail, they are poised to modify the 5′ end of the nascent transcript as soon as it emerges from the polymerase.
- 4. The cap protectsthe 5′ end of the primary RNA transcript from attack by ribonucleases and is recognized by eukaryotic initiation factors involved in assembling the ribosome on the mature mRNA prior to initiating translation
- 5. • The 5′-methylcap signals the 5′ end of eukaryotic mRNAs, and this landmark helps the cell to distinguish mRNAs from the other types of RNA molecules present in the cell. • For example, RNA polymerases I and III produce uncapped RNAs during transcription, in part because these polymerases lack tails. In the nucleus, the cap binds a protein complex called CBC (cap-binding complex), which, as we discuss in subsequent sections, helps the RNA to be properly processed and exported. The reactions that cap the 5′ end of each RNA molecule synthesized by RNA polymerase II
- 6. POLY-A TAIL • Thepoly-A tail is a long chain of adenine nucleotides that is added to a messenger RNA (mRNA) molecule during RNA processing to increase the stability of the molecule. • Immediately after a gene in a eukaryotic cell is transcribed, the new RNA molecule undergoes several modifications known as RNA processing. • These modifications alter both ends of the primary RNA transcript to produce a mature mRNA molecule. The processing of the 3' end adds a poly-A tail to the RNA molecule. • First, the 3' end of the transcript is cleaved to free a 3' hydroxyl. Then an enzyme called poly-A polymerase adds a chain of adenine nucleotides to the RNA.
- 7. • This process,called polyadenylation, adds a poly-A tail that is between 100 and 250 residues long. • The poly-A tail makes the RNA molecule more stable and prevents its degradation. • Additionally, the poly-A tail allows the mature messenger RNA molecule to be exported from the nucleus and translated into a protein by ribosomes in the cytoplasm. • Cleavage and polyadenylation specificity factor (CPSF). • Cleavage stimulatory factor or cleavage stimulation factor (CstF or CStF) . • Poly(A) polymerase (PAP) • Poly(A)-binding protein (PAB or PABP
- 8. The hnRNA runs5' 3' from the top left to lower right, where transcription terminates Upon recognition of the AAUAAA signal, the 3' end is removed by an enzyme complex [CFI, II] at the cut site. Another enzyme complex [PA] synthesizes a poly(A) tail and ligates it to the free 3' of the pre-message Post-transcriptional processing of mRNA; polyadenylation of 3' tail
- 9. RNA Splicing • Theprotein coding sequences of eukaryotic genes are typically interrupted by noncoding intervening sequences (introns). • An intron is a long stretch of noncoding DNA found between exons (or coding regions) in a gene. • Genes that contain introns are known as discontinuous or split genes as the coding regions are not continuous. Introns are found only in eukaryotic organisms. • It is considered as “junk DNA”, introns likely play an important role in regulation and gene expression.
- 10. EXONS • The sectionsof DNA (or RNA) that code for proteins are called exons. • Following transcription, new, immature strands of messenger RNA, called pre-mRNA, may contain both introns and exons. • The pre-mRNA molecule thus goes through a modification process in the nucleus called splicing during which the noncoding introns are cut out and only the coding exons remain.
- 11. • During transcriptionRNA polymerase copies the entire gene, both introns and exons, into the initial mRNA transcript known as pre-mRNA or heterogeneous nuclear RNA (hrRNA). • As introns are not transcribed, they must then be removed before translation can occur. • The excision of introns and the connection of exons into a mature mRNA molecule occurs in the nucleus and is known as splicing. • Introns contain a number of sequences that are involved in splicing including spliceosome recognition sites. • These sites allow the spliceosome to recognise the boundary between the introns and exons. • The sites themselves are recognised by small nucleolar ribonucleoproteins (snRNPs). • There are a number of snRNPs involved in mRNA splicing which combined create a spliceosome.
- 12. Splicing occurs inthree steps: • Cleavage of the phosphodiester bond between the exon and the GU at the 5′ end of the intron. One snRNP (U1) contains a complementary sequence to the 5′ splice site and binds there to initiate splicing. • Formation of a lariat or loop structure. The free 5′ end of the intron connects to a branch site, a conserved sequence near the 3′ end of the intron. A second snRNP (U2) binds to the branch site and attracts U1 to initiate the lariat. The lariat is then formed by a phosphodiester bond between the free 5′ G and an A at the branch site. • Cleavage of the phosphodiester bond between the second exon and the 3′ AG of the intron.
- 13. The RNA splicingreaction
- 14. • Introns rangein size from about 10 nucleotides to over 100,000 nucleotides. Picking out the precise borders of an intron is very difficult. • Intron sequence removal involves three positions on the RNA: the 5′ splice site, the 3′ splice site, and the branch point in the intron sequence that forms the base of the excised lariat. • In pre-mRNA splicing, each of these three sites has a consensus nucleotide sequence that is similar from intron to intron, providing the cell with cues on where splicing is to take place.
- 15. RNA Splicing bythe Spliceosome • RNA splicing is performed largely by RNA molecules instead of proteins. RNA molecules recognize intron-exon borders and participate in the chemistry of splicing. • These RNA molecules are relatively short (less than 200 nucleotides each), and there are five of them (U1, U2, U4, U5, and U6) involved in the major form of pre-mRNA splicing Known as snRNAs (small nuclear RNAs), • each is complexed with at least seven protein subunits to form a snRNP (small nuclear ribonucleoprotein). • These snRNPs form the core of the spliceosome, the large assembly of RNA and protein molecules that performs pre-mRNA splicing in the cell.
- 16. • RNA splicingis catalyzed by an assembly of snRNPs (“snurps”) plus other proteins which together constitute the spliceosome. • The spliceosome recognizes the splicing signals on a pre- mRNA molecule, brings the two ends of the intron together, and provides the enzymatic activity for the two reaction step. • The branch-point site is first recognized by the BBP (branch-point binding protein) and U2AF, a helper protein. • In the next steps, the U2 snRNP displaces BBP and U2AF and forms base pairs with the branch-point site consensus sequence, and the U1 snRNP forms base-pairs with the 5′ splice junction . • At this point, the U4/U6•U5 “triple” snRNP enters the spliceosome. • In this triple snRNP, the U4 and U6 snRNAs are held firmly together by base-pair interactions and the U5 snRNP is more loosely associated. • Several RNA-RNA rearrangements then occur that break apart the U4/U6 base pairs (as shown, the U4 snRNP is ejected from the splicesome before splicing is complete) and allow the U6 snRNP to displace U1 at the 5′ splice junction . • Subsequent rearrangements create the active site of the spliceosome and position the appropriate portions of the pre-mRNA substrate for the splicing reaction to occur. • Each splicing event requires additional proteins, some of which hydrolyze ATP and promote the RNA-RNA rearrangements.
- 17. ALTERNATIVE SPLICING • Alternativesplicing, or alternative RNA splicing, or differential splicing, is an alternative splicing process during gene expression that allows a single gene to code for multiple proteins. • In this process, particular exons of a gene may be included within or excluded from the final, processed messenger RNA (mRNA) produced from that gene,means the exons are joined in different combinations, leading to different (alternative) mRNA strands. • The proteins translated from alternatively spliced mRNAs will contain differences in their amino acid sequence and, often, in their biological functions.
- 18. • Alternative splicingoccurs as a normal phenomenon in eukaryotes, where it greatly increases the biodiversity of proteins that can be encoded by the genome; • in humans, ~95% of multi-exonic genes are alternatively spliced. • There are numerous modes of alternative splicing observed, of which the most common is exon skipping. • In this mode, a particular exon may be included in mRNAs under some conditions or in particular tissues, and omitted from the mRNA in others. • Eg: gene encoding the thyroid hormone calcitonin was found to be alternatively spliced in mammalian cells. • The Adenovirus produces five primary transcripts early in its infectious cycle, prior to viral DNA replication, and an additional one later, after DNA replication begins. • The "record-holder" for alternative splicing is a D. melanogaster gene called Dscam, which could potentially have 38,016 splice variants.
- 19. Five basic modesof alternative splicing are generally recognized •Exon skipping or cassette exon: in this case, an exon may be spliced out of the primary transcript or retained. This is the most common mode in mammalian pre-mRNAs. •Mutually exclusive exons: One of two exons is retained in mRNAs after splicing, but not both. •Alternative donor site: An alternative 5' splice junction (donor site) is used, changing the 3' boundary of the upstream exon.
- 20. • Alternative acceptorsite: An alternative 3' splice junction (acceptor site) is used, changing the 5' boundary of the downstream exon. • Intron retention: A sequence may be spliced out as an intron or simply retained. This is distinguished from exon skipping because the retained sequence is not flanked by introns. If the retained intron is in the coding region, the intron must encode amino acids in frame with the neighboring exons, or a stop codon or a shift in the reading frame will cause the protein to be non-functional. This is the rarest mode in mammals.
- 22. • Alternative splicingis a major diversification mechanism in the human transcriptome and proteome. Several diseases, including cancers, have been associated with dysregulation of alternative splicing. Thus, correcting alternative splicing may restore normal cell physiology in patients with these diseases. • Trans-splicing is a special form of RNA processing where exons from two different primary RNA transcripts are joined end to end and ligated. • It is usually found in eukaryotes and mediated by the spliceosome, although some bacteria and archaea also have "half-genes" for tRNAs.