CMB2001 Lecture 8 Post-transcriptional Gene Expression (PDF)

CMB2001 Lecture 8 Post-transcriptional control of gene expression –Part 2 Dr Shiney Mathew [email protected] Join the Vevox session Go to vevox.app Enter the session ID: 174- 573-785 Or scan the QR code Recap 1. Transcription cont...

CMB2001 Lecture 8 Post-transcriptional control of gene expression –Part 2 Dr Shiney Mathew [email protected] Join the Vevox session Go to vevox.app Enter the session ID: 174- 573-785 Or scan the QR code Recap 1. Transcription control Regulation is possible at each step 2. RNA processing control Pre-mRNA undergoes processing events to form mRNA 3. Translational control RNA processing events -Capping -Splicing Alternative splicing Alternative splicing increases 4. Protein activity control coding capacity Disease associated with splicing defects Primary transcript Primary Central transcript processing Central dogma dogma of processing of gene gene expression expression The unfinished messenger RNA or precursor messenger RNA is known as pre-mRNA Capping Splicing - Alternative splicing Polyadenylation Editing mRNA localisation Polyadenylation DNA 5’ cap AAUAAA Nascent RNA endonuclease cleavage Addition of As by polyA polymerase 5’ cap AAUAAA AAAAAAAAAAAAAA(A) n OH 3’ polyadenylated mRNA Polyadenylation polyA site 5’ USE AAUAAA G/U 3’ Conserved AAUAAA 10-35 nucleotides upstream of the poly(A) site. G/U or U rich tract just downstream of poly(A) site U-rich upstream element “USE” Polyadenylation polyA site CPSF CstF 5’ USE AAUAAA G/U 3’ 5’ USE AAUAAA AAAAAAAAA…… PAP Proteins required for polyadenylation bind these sequences: Cleavage and polyadenylation specificity factor (CPSF) binds AAUAAA Cleavage stimulatory factor (CstF) binds G/U (Cleavage factors I and II (CFI, CFII)) Poly(A) polymerase Functional significance of the poly A tail Central dogma of gene expression All mRNAs has 3’ poly(A) tail (a few exceptions e.g. histones) Approx 250 nucleotides long Bound by poly A-binding protein Enhances export of RNA Stabilises the 3’ end of the mRNA Enhances translation of mRNA Things to think about poly A tail Unusual structure, long single nucleotide sequences are rare Polyadenylation linked to transcription Important structure for mRNA stability, production and function The poly A tail is a protein-binding element Primary transcript processing Central dogma of gene expression The unfinished messenger RNA or precursor messenger RNA is known as pre-mRNA Capping Splicing - Alternative splicing Polyadenylation Editing mRNA localisation What is RNA editing? Central dogma of gene expression RNA editing nucleotide alterations which result in different or additional nucleotides in the mature RNA Occurs in the 3 major classes of RNA mRNA, tRNA, ribosomal RNA (rRNA) Two classes of editing: insertion/deletion modification (e.g. A to I, C to U, U to C) Change the coding sequence and/or properties of mRNAs Significance of RNA Central dogma of gene editing in medicine and development expression Disease Atherosclerosis Brain function Human higher brain function and depression Development Drosophila Parasites Trypanosoma Leishmania and Trypanosoma mitochondria - potential target for drugs Base modification Central dogma of gene expression ‘marked nucleotide’ NB – reversible! (NB don’t confuse with m5C in DNA, which is a common epigenetic mark!) Altered identity (I read as G, pairs with C) Effects ofofmRNA Central dogma editing gene expression creation of creation of start codons creation of new open stop codons by U insertion reading frames by by U insertion nucleotide insertion mRNA AUG STOP AAAAAAAAA removal of stop codons by base conversions creation of start codons by C to changes in encoded creation of stop amino acids and codons by C to U changes splice site choice by U changes base conversion RNA Editing by Deamination Central dogma of gene expression Enzymatic Deamination Inosine recognised as guanosine Therefore equivalent of an A to G change Cytidine deamination: apoB pre-mRNA editing Central dogma of gene expression AUG CAA UAA mRNA Intestine (editing) Liver (no editing) AUG UAA UAA AUG CAA UAA Translation Translation ApoB-48 ApoB-100 LDL-receptor binding Editing carried out by the APOBEC-1 enzyme (linked to cholesterol control, cancer development and inhibition of viral replication) Both forms circulate in blood but have different functions The long form major component of very low density lipoproteins linked to atherosclerosis A to I editing in the Q/R Site of Glutamate Receptors Central dogma of gene expression L-glutamate major excitatory neurotransmitter Editing yields decrease in Ca2+ permeability of channels containing the ‘R’ version Glu Extent of editing varies in different parts of the Q Na+ body Ca2+ Editing carried out by ADAR2 (adenosine postsynaptic deaminase acting on RNA). neuron Mutations in the mouse ADAR2 gene lead to seizures, post-natal death, neurodegeneration in R Na+ Ca2+ the hippocampus. Glu Things to think about RNA editing After transcription, individual nucleotides can be modified or changed Can result in markers being put on the RNA These result in changes in protein-binding, leading to changes in the property of the RNA Can also result in a change in the sequence Editing can also occur on ncRNAs and is essential for their function Export of mRNA 1. Transcription control 2. RNA processing control Export of mRNA 3. Translational control from nucleus to cytoplasm 4. Protein inactivity control The nuclear pore Different pathways mediate the export of different classes of RNA  Primary transcript processing Central dogma of gene expression The unfinished messenger RNA or precursor messenger RNA is known as pre-mRNA Capping Splicing - Alternative splicing Polyadenylation Editing mRNA localisation Asymmetric mRNA localisation Why localise mRNA? Localised protein synthesis Generate cell polarity bicoid, nanos in Drosophila embryo Drosophila neuroblasts ß-actin in fibroblasts Ash1 mRNA in yeast Prevents expression in the wrong place e.g myelin basic protein Promotes efficiency of subsequent protein targeting ß-actin in fibroblasts, perinuclear metallothionein Local control of translation e.g. synapses Localisation of mRNAs during Drosophila development Localisation of dendritic mRNAs Diffusion based-localisation mRNAs freely diffuse in the cytoplasm and are locally entrapped by anchor proteins Active transport based-localisation mRNA recognized by specific trans-acting factors in the nucleus cytoplasmic factors ensure transport along a polarized cytoskeleton Things to think about RNA export and localisation Many RNAs need to be exported to the cytoplasm Many mRNAs are localised asymmetrically in the cytoplasm This is required for localised translation – essential for many processes in the cell Active or passive transport is used to localise the mRNAs Summary Polyadenylation addition of Poly A tail RNA editing insertion/deletion base modification Nuclear export Different mechanisms for different classes of RNA Localisation of mRNA many RNAs are asymmetrically localised achieved by diverse mechanisms essential for cell differentiation and function