Control of gene expression Part II 2023.pdf

Post-transcriptional control of gene expression Posttranscriptional control Overview of mRNA Processing in Eukaryotes üProcessing of pre-mRNA is co-transcriptional Regulation of pre-mRNA processing Ø Alternative RNA splicing Ø cleavage at alternative polyadenylation site These may create dif...

Post-transcriptional control of gene expression Posttranscriptional control Overview of mRNA Processing in Eukaryotes üProcessing of pre-mRNA is co-transcriptional Regulation of pre-mRNA processing Ø Alternative RNA splicing Ø cleavage at alternative polyadenylation site These may create different proteins Effect of Alternative Splicing on Gene function Calcium/calmodulin-dependent kinase IId (CaMKIId) : A serine/threonine kinase which is activated by increased calcium concentration in cells Differentiatial splicing of the pre-mRNA for kinase results in production of three different kinases localized in different locations in the cells and kinasing different substrates Alternative RNA splicing • Alternative RNA splicing can produce different forms of a protein from the same gene. • One form may be functional, and the other form is non-functional • Two forms may have different localization signal • Different forms may have different affinities for substrate or ligand binding • RNA splicing can be regulated either negatively, by a regulatory molecule that prevents the splicing machinery from gaining access to a particular splice site on the RNA, or possibly by a regulatory molecule that helps direct the splicing machinery to an otherwise overlooked splice site. Alternative cleavage site and PolyA addition during B-cell activation • IgG is membrane bound in non-activated (naïve) B-cells • Upon activation B cells secretes IgG • Membrane bound immunoglobulin (mIg) and secreted immunoglobulin are produced from the same gene by using different sites for cleavage and polyadenylation. Regulation of the site of RNA cleavage and Poly-A addition • • • * * * * * • • • • • Figure 7-59 Molecular Biology of the Cell • • IgG pre-mRNA has 2 cleavage/polyadenylation sites IgG pre-mRNA also has 2 stop codons (*, *); one inside the intron and the other after the last exon. In naïve B cells long IgG transcript is produced due to utilization of second cleavage site This long transcript has 2 exons (blue and green) and one intron (yellow) After splicing the matured mRNA uses the second stop codon to make a larger protein The longer form of IgG has a terminal hydrophobic region that binds to cell surface membrane In activated B cells shorter IgG transcript is produced due to utilization of the first cleavage/polyadenylation site. This shorter transcript has 1 exons (blue), but the intron (yellow) acts as an intron, because there is no exon after this. After splicing the matured RNA uses the first stop codon and make a smaller protein The shorter form of IgG lacks this hydrophobic region, it can not bind to the cell membrane, thus secreted RNA editing • • • RNA editing: Changes in nucleotide in mRNA (not due to gene mutation) that alters protein sequence In higher vertebrates, RNA editing is much rare, and mostly in single base changes by enzymatic modification RNA editing is widespread in the mitochondria of certain protozoa and plants, and in chloroplasts A to I conversion A to I conversion in mammalian pre-mRNA coding for a transmitter gated ion channel in the brain Nucleus Pre-mRNA mRNA splicing A Cytosol CAA (Q) matured-mRNA mRNA-editing Change of Glutamine (Q) to Arginine (R), alter Ca2+ permeability through the channel I CIA CGA (R) edited-mRNA ADAR mutants are lethal ADAR: Adenosine Deaminase Acting on RNA Figure 7-101 Molecular Biology of the Cell (© Garland Science 2008) Glutamate receptor RNA editing and Amyotrophic Lateral Schlerosis (ALS) • Glutamate causes excitation of the synaptic neurons • Glutamate is taken up by the Glutamate receptors (GluR) • Un-edited GluR allows more Ca2+ into neurons • Increased levels of Ca2+ damage mitochondria and caused cell apoptosis • editing decreased the levels of Ca2+ transport and maintains cellular homeostasis • ADAR mutation reduced edited Q to R editing and caused ALS C to U conversion • • Apolipoprotein B (ApoB) is present in low-density lipoprotein (LDL) particles that carry cholesterol and transferred to cell via LDLreceptor ApoB gene in mammals is one of the example of RNA editing. In the liver, ApoB gene is transcribed into a longer mRNA, which encodes a protein with two domains (green domain binds to lipid and the orange domain binds LDL receptor). In the intestine, the CAA codon of ApoB is edited to UAA [a stop codon] so the protein [ApoB-48] can bind lipid but not with LDL receptor APOBEC: Apolipoprotein B RNA Editing Catalytic subunit Cellular iron homeostasis Fe Fe Transferrin receptor Transferrin Extracellular Cytosol Cell membrane Ferritin: iron-binding protein required for iron storage Transferrin receptor: required for iron uptake Transferrin: carries iron Fe Ferritin FeFe Fe Fe Low cellular iron levels Ferritin Transferrin receptor High cellular iron levels Ferritin Transferrin receptor Regulation of ferritin and transferrin receptor expression by iron Aconitase: an iron binding protein, binds the stemloop structure of RNA in the absence of iron Ø Ø Ø Ø While cytosolic aconitase is an iron-binding protein. In the absence of iron it binds with RNA but in the presence of iron it can not bind RNA When iron is low in the cell aconitase binds with the 5’ and 3’ UTR regions of the ferritin and transferrin mRNAs, respectively. Aconitase binding inhibits ferritin translation, so less ferritin is produced. However, binding of aconitase to the 3’UTR of transferrin makes this RNA stable that increases production of transferrin In the presence of iron ferritin is made but no transferrin is produced and uptake of iron is reduced Figure 7-111 Molecular Biology of the Cell (© Garland Science 2008) Global control of protein synthesis by eIF2 phosphorylation Deprivation of growth factors, nutrients, infection by viruses, and sudden increases in temperature Activate protein kinase eIF2 phosphorylates Inhibition of protein synthesis Figure 7-67 Molecular Biology of the Cell Internal ribosome entry sites provide opportunities for translational control Two mechanisms of translation initiation Ø Some viruses use IRES to synthesize their own protein while blocking the normal initiation of the host protein synthesis Ø These viruses truncated eIF4G so it can not bind to eIF4E (5’cap structure) Ø However, truncated eIF4G binds on IRES sites on the viral RNA and initiates translation of the viral proteins IRES; internal ribosome entry site Figure 7-68 Molecular Biology of the Cell Control of gene expression by mRNA stability Figure 7-69 and 70 Molecular Biology of the Cell • A critical threshold of PolyA tail length induces rapid 3’-to-5’ degradation • Decapping and degradation from 5’-to-3’ end is followed • The same two features of an mRNA molecule e.g., its 5’ cap and the 3’ poly-A tail are used both in both translation initiation and deadenylation-dependent mRNA decay • Loss of poly-A binding protein facilitates deadenylase to shorten poly-A tail Noncoding RNAs play multiple roles in controlling gene expression § Only a small fraction of DNA codes for proteins, and a very small fraction of the non-protein-coding DNA consists of genes for RNA such as rRNA and tRNA § A significant amount of the genome may be transcribed into noncoding RNAs (ncRNAs) § There are various types of ncRNAs. These include a) small nuclear RNA (snRNA, ~150 b), b) long nuclear RNA (lncRNA, >200 b), c) small nucleolar RNA (snoRNA), d) microRNA (miRNA) , e) small interfering RNA (siRNA), and piwi-interacting RNA (piRNA) § ncRNA performs various functions, e.g., gene specific transcription, regulating basal transcription machinery, splicing, translation, mRNA degradation, epigenetic modification, genomic imprinting, X-chromosome inactivation, and telomere protection Small non-coding RNA 1. Micro RNAs (miRNAs) 2. Small interfering RNAs (siRNAs) 3. Piwi interacting RNAs (piRNAs) How they work? Figure 7-74 Molecular Biology of the Cell Regulation of gene expression by miRNA RISC: RNA induced silencing complex Argonaute: An essential component of the RISC complex Human Argonaute protein Figure 7-75 and 76 Molecular Biology of the Cell) Mechanism of action of miRNAs and siRNAs • Over 1000 different miRNAs are produced from the human genome • Once made, miRNA base pair with specific mRNAs and regulate their translation or stability • The miRNA precursors are synthesized by RNA polymerase II. These are capped and polyadenylated • Then they processed by Dicer to short double stranded RNA (~23 nucleotides in length • This short double stranded RNA assembled with a set of proteins to form RNAinducing silencing complex (RISC). One strand of RNA is lost or degraded. Thus, RISC complex has one RNA strand • Once formed RISC seeks out its target mRNA by searching complementary nucleotide sequence • Once the target mRNA is bound with the miRNA or siRNAs the target RNA is degraded if the base-pairing is extensive • If the base pairing is not extensive, the target mRNA translation is inhibited Functions of miRNAs and siRNAs miRNA: • The major function of miRNA is to regulate gene expression • One miRNA can target a whole set of different mRNAs • Multiple miRNA can target different regions of a single mRNA and ultimate effect is combinatorial • miRNAs are shorter than gene regulatory proteins thus occupies smaller spaces in the genome siRNA: • Major function of siRNAs to degrade foreign double stranded RNAs, like invading viral RNAs • Play important role in plant by protecting from viral infection. • In plant, siRNAs are amplified by RNA-dependent RNA polymerase (RDRP) and transferred from one cell to another to provide overall immunity to virus attack • Control transposable element by degrading double stranded RNA generated from these elements • It has been used as a powerful experimental tool to dissect gene functions. Gene silencing by siRNA Dicer: cleaves double stranded RNA to form small interfering RNAs (siRNAs) RITS: RNA-induced Transcriptional Silencing Figure 7-77 Molecular Biology of the Cell Noncoding RNA in bacteria CRISPR RNAs • • • • • • • CRISPR (Clustered regularly interspersed short palindromic repeat) locus has been found in many bacterial genome CRISPR RNAs (CrRNAs) are formed to attack invading viral DNA or other foreign DNAs CrRNAs bind with Cas protein, a class of Argonaute and Piwi proteins Small pieces of viral DNA integrated into a specific locus in bacterial genome during previous infection to produce Cr locus and inherited through following generations During future invasion of a similar virus, CrRNAs those are already present in the bacteria, target these viral DNA and destroy the virus Thus, CRISPR system creates bacterial immunity to virus attack This system is an important genetic tool used in laboratories for gene editing. Figure 7-78 Molecular Biology of the Cell Long Noncoding RNAs (lncRNA) • • • • • lncRNAs are defined as RNAs that are longer than 200 nucleotides and do not code for proteins lncRNAs are transcribed by RNA PolI and have 5’ caps and poly-A tails Examples are telomerase RNA, Xist RNA, and others Expression levels are very low lncRNAs perform many functions; • Acts as scaffold molecule holding together several proteins for their function • Has ability to serve as the guide sequences, binding to specific RNA or DNA targets • Can bind with nascent RNA to change the splicing pattern • Acts as sponge RNA to to neutralize many miRNAs Figure 7-79 Molecular Biology of the Cell Similarities and differences between mRNA and lnc RNA mRNA lncRNA Tissue-specific expression Tissue-specific expression Form secondary structure Form secondary structure Undergo post-transcriptional processing, e.g., 5’ cap, polyadenylation, splicing Undergo post-transcriptional processing, e.g., 5’ cap, polyadenylation, splicing Important roles in disease and development Important roles in disease and development Protein coding transcripts Non-protein coding, regulatory function Well-conserved between species Poorly conserved between species Present in both nucleus and cytoplasm Predominantly nuclear, others nuclear and/or cytoplasm Total 20-24,000 mRNAs Currently, 30,000 Lnc transcripts, predicted 3-100 fold of mRNA in number Expression level: low to high Expression level: very low to moderate What did we learn from this section? • Regulation of gene expression at the levels of RNA processing; trans-splicing and alteration of poly-A addition site • RNA editing and generation of two isoforms of ApoB and glutamate receptor with different functions • Translational control of gene expression; specific control and global control • Internal ribosome entry site and its role in viral replication • Control at RNA stability • Types of non-coding RNAs and their functions; miRNA, siRNA, piRNA, crRNA, and lncRNA