POST TRANSCRIPTIONAL MODOFICATION.pptx

POST TRANSCRIPTIONAL MODOFICATIONS

1. POST TRANSCRIPTIONAL MODOFICATION (Splicing Mechanism) Dr. P. Suganya Assistant Professor Sri Kaliswari College (Autonomous), SIvakasi

2. Prokaryotes: RNA transcribed from DNA template and used immediately in protein synthesis Eukaryotes : Primary transcript ( hn RNA) must undergo certain modifications to produce mature mRNA (active form) for protein synthesis.

3. “Post-transcriptional modification is a set of biological processes common to most eukaryotic cells by which an primary RNA 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.”

4. OVERVIEW Transcription Genetic information from DNA is copied into messenger RNA (mRNA). In this process, mRNA is synthesized from the 5’ end to the 3’ end. The initial transcript is known as heterogeneous nuclear RNA ( hnRNA ) or pre-mRNA.

5. Gene expression from DNA, the genetic sequence, is transcribed into the RNA (transcription): Transcription of genetic information is the first step in gene expression and is the process through which a coding region of DNA (double-stranded structure) is used as a template for the synthesis of messenger RNA (mRNA). The mature mRNA is translated into amino acids, forming proteins (translation) with the help of ribosomal RNA and transfer RNA ( tRNA ). This image shows transcription without post-transcriptional modifications of the RNA.

6. Modifications Primary transcripts, or immediate products of transcription, undergo alterations to become biologically functional. mRNA: Prokaryotes: Most primary mRNAs have no modifications. Eukaryotes: Synthesized transcript of mRNA (or hnRNA ) undergoes processing before leaving the nucleus. Addition of 5’ cap Addition of 3’ poly-A tail Splicing

7. Modification of hnRNA produces mature mRNA, which is transported to the cytoplasm through nuclear pores. In some cases, RNA editing occurs with base changes, creating a sequence different from that copied from the DNA. A different mRNA sequence produces a different protein; this varies from the old hypothesis of  “one gene–one polypeptide.” Transfer RNA ( tRNA ) and ribosomal RNA ( rRNA ):  Structural molecules that are not translated Both have pre- tRNAs and pre- rRNAs that undergo processing.

8. Summary of post-transcriptional modifications of hnRNA into a mature mRNA: The addition of the 5’ cap and the 3’ poly-A tail and splicing (removal of the intervening sequences or introns )

9. Addition of the 5′ Cap and 3′ Poly-A Tail 5′ cap 7-Methylguanosine ( methylated guanylyl residue) is added to the 5’ end of hnRNA via: Removal of the leading phosphate group at the 5’ terminal by RNA triphosphatase Transfer of guanosine monophosphate (GMP) from the guanosine triphosphate group by guanylyl transferase Methylation of guanine by guanine-7-methyltransferase (methyl group from  S - adenosylmethionine (SAM)) Functions: Prevents exonuclease degradation Recognition sequence for translation

10. 3′ Poly-A tail 50 to 250 adenylyl residues (AMP) are added to the 3’ end of hnRNA via: Cleaving of about 20 nucleotides downstream from an AAUAA recognition sequence Addition (and extension up to 250 nucleotides) of poly-A tail (generated from ATP) by poly-A polymerase Function: Prevents degradation in the cytosol by 3′ exoribonucleases Stabilizes mRNA

11. Post-transcriptional modifications of RNA: The 5’ cap (7-methylguanosine) and 3’ poly-A tail modifications prevent degradation of the mRNA in the cytosol .

12. Heterogeneous Nuclear RNA Splicing Exons and introns Heterogeneous nuclear (pre-mRNA) contains: Coding sections called exons (expressed sequences) Noncoding sections called introns (intervening sequences) Processing: hnRNA needs processing (splicing) to produce the mRNA carrying the proper coding sequences. Occurs in most eukaryotic genes,   most commonly on mRNA

13. Pre-mRNA exons and introns with an overview of splicing (from top to bottom): Pre-mRNA transcript contains exons and introns . Interactions of the transcript with small nuclear ribonucleoproteins and other proteins form a spliceosome at certain junctions of the transcript. Cuts are made at the splice sites, and the intron is released. Spliced RNA now only has exons , which contain the coding sequence.

14. Splicing Removal of introns from the hnRNA /pre-mRNA, while linking the exons to form the mature mRNA Process involves the hnRNA and additional components: Small nuclear ribonucleoproteins (which are made up of small nuclear RNAs ( snRNAs ) and proteins) Other binding proteins

15. Junctions where splicing reaction occurs:Splice sites:  Areas where cuts are made between the exon and intron   Base sequences identify these sites, one at the 5’ side (beginning of the intron ) and the other at the 3’ side (end of the intron ). 5’ site/donor splice site: invariant GU 3’ site/acceptor splice site: invariant AG  Branch site: located upstream from 3’ site

16. Mechanism Small nuclear ribonucleoproteins recognize the splice sites and branch site owing to the base sequences on the hnRNA . hnRNA , small nuclear ribonucleoproteins , and other proteins combine to form the spliceosome .  The spliceosome complex makes a cut on the 5’ donor splice site (occurs via a nucleophilic attack by an adenylyl residue in the branch site). The now free 5’ terminus of the intron links to the branch site, forming a loop, or lariat, structure. The 3’ splice site is recognized, and the second cut occurs there. Release of the lariat follows, and the 2 exons are joined to form the mature RNA Occurs simultaneously with the 5’ cap and 3’ poly-A tail hnRNA modifications 

17. Alternative splicing Differential splicing of one hnRNA sequence Mechanisms: Exons are selectively included or excluded. Alternative 5′ donor or 3′ acceptor sites are used. Polyadenylation sites can differ.

18. Up to 95% of multi- exon genes undergo alternative splicing (AS) to encode proteins with different cellular functions. AS is a rapidly responsive regulation step needed for fine-tuning protein synthesis and thereby determining cell phenotypes and proliferation rates. Approximately 15% of hereditary diseases and cancers are reported to be associated with AS. Different combinations of exons can lead to different related proteins being created from the same hnRNA : Immunoglobulin molecules (genes for heavy chains have exons related to individual subtypes) Tropomyosin variants in muscle Dopamine receptors in the brain (D2 receptors with 2 isoforms )

19. Examples of alternative splicing: Protein A: Exons 1–5 were joined after splicing of introns . Proteins B and C: An exon was selectively excluded to form a different protein

20. References https://en.wikipedia.org/wiki/Post-transcriptional_modification#:~:text=Post%2Dtranscriptional%20modification%20or%20co,the%20nucleus%20and%20perform%20any