2. FunMed - RNA Transcription Mullaney.pptx

FunMed Lecture – 3rd October 2023 Transcription and RNA processing Dr Ian Mullaney Senior Lecturer in Medical Sciences QMUL Malta Dr Ian Mullaney Learning Objectives 1. Understand the importance of gene expression for cellular identity. 2. Appreciate the crucial role mRNA synthesis plays in this....

FunMed Lecture – 3rd October 2023 Transcription and RNA processing Dr Ian Mullaney Senior Lecturer in Medical Sciences QMUL Malta Dr Ian Mullaney Learning Objectives 1. Understand the importance of gene expression for cellular identity. 2. Appreciate the crucial role mRNA synthesis plays in this. 3. Be able to describe the basic steps that create lead to translatable mRNA in the cytoplasm. 4. Recognise that aberrant gene expression is a major cause of disease. Dr Ian Mullaney Learning Objective 1 1. Understand the importance of gene expression for cellular identity. Dr Ian Mullaney What is gene expression? Gene expression is the process whereby information contained within the genetic components in the nucleus of the cell (genome) is utilised to produced specific physical traits within the individual (phenome). Each of these genetic components (genes) has the potential to produce one or more proteins that contribute to the overall phenotypic make up of an individual. For example, the genetic potential for eye colour when expressed results in a myriad of physically present colours. One gene OCA2, found on chromosome 12, codes for brown eye pigment. If one or both parents have a fully functional gene, their offspring will have brownpigmented eyes. However if both parent have faulty OCA2 expression, non-pigmented pale usually blue eyes result. Dr Ian Mullaney To put things into perspective, the human genome he structure of a typical gene? The component parts of a typical eukaryotic gene include: 1. Exon – Sections of DNA that comprise a coding region of a gene that contains the information required to encode a protein. 2. Intron - A stretch of DNA that resides within a gene but does not code for amino acids that make up the protein encoded by that gene. 3. Promotor region - DNA sequences that define where transcription of a gene by RNA polymerase begins. Promoter sequences are typically located directly upstream or at the 5' end of the Dr Ian Mullaney elationship between nucleic acids and proteins – The Central Dogma The central dogma describes how information contained within the DNA of the genome is used to produce physical structures necessary for life. Two processes are involved: 1. Transcription: the process of converting the genetic information existing as DNA into an intermediary or adapter molecule known as messenger RNA (mRNA). 2. Translation: The use of various RNA molecules and enzymes to convert the information contained within the mRNA to proteins. mRNA serves as an intermediate in the flow of information from DNA to protein, the primary functional molecule in the Dr Ian Mullaney cell. Thus stable nucleotide information is DNA makes RNA makes protein e molecular structure of RNA (Ribonucleic acid) Like DNA, RNA is composed of mononucleotides joined by phosphodiester bonds. However, the sugar component is ribose instead of deoxyribose and the base thymine is replaced by uracil Unlike DNA, the RNAs exist as single strands although some intramolecular folding can occur Dr Ian Mullaney trogenous bases – derived from nucleotides. Oxyribonucleotides (RNA) V Deoxyribonuceotides (DNA) e molecular structure of RNA (Ribonucleic acid) Dr Ian Mullaney Dr Ian Mullaney Learning Objective 2 2. Appreciate the crucial role mRNA synthesis plays in gene expression for cellular identity. Dr Ian Mullaney Transcription – The production of mRNA from a genomic DNA template. There are numerous different types of RNA molecules within the cell, all having distinct cellular functions. The three most important with respect to the transfer of genetic information to active protein molecules are (i) messenger RNA (mRNA), (ii) transfer RNA (tRNA) and (iii) ribosomal RNA (rRNA). This lecture will concentrate on mRNA, the adapter molecule which is formed as a result of DNA transcription. Production of useable mRNA from the DNA template depends on 2 processes. Firstly a primary transcript is produced in the cell nucleus then that transcript undergoes further Mullaney processing allowing the finished mRNADrtoIanbe a Transcription – Synthesis of mRNA. Transcription is mediated by the enzyme RNA polymerase. There are 3 types of RNA polymerase found in eukaryotic cells. These are termed: RNA polymerase I: which synthesises the large rRNA molecules (28S, 18S, 5.8S) RNA polymerase II: which synthesis the primary mRNA transcripts RNA polymerase III: which produces the small RNAs. These include tRNA’s and the small 5S rRNA. We will concentrate on RNA polymerase II In addition to RNA polymerase, there are a number of associated transcription factors that bind to the DNA template. The binding of different factors determines which genes are to be transcribed. In addition, since eukaryotic DNA exists in a complex with histone proteins (nucleosome), temporary DNA dissociation must occur for transcription to occur. Dr Ian Mullaney Transcription factors and promoter regions. Transcription factors and promotor regions help determine which gene gets transcribed. The most important promotor regions are: i. The CAAT box: found 70 to 80 nucleotides from the start site. Transcription factor CTF binds here. ii. The TATA or Hogness box: found 25 bases from the site of transcription Transcription factor TFIID binds here. iii. The GC box: found anywhere in the regions before transcription. Transcription factor SP1 binds here. Dr Ian Mullaney Transcription – 1. Initiation In order for transcription to occur, there is local unwinding of the double stranded DNA close to the gene of interest. RNA polymerase II then binds to a promotor sequence. Recruitment of transcription factors occurs (termed the preinitiation complex) which complex to Pol II and transcription begins. 3’ 5’ 5’ 3’ Dr Ian Mullaney Transcription – 2. Elongation RNA polymerase II uses incoming ribonucleotides to form the new mRNA strand. It does this by catalysing the formation of phosphodiester bonds between adjacent ribonucleotides, using complementary base pairing (A to U, T to A, C to G and G to C). During transcription, only the 3’5’ DNA strand is transcribed. This is the antisense or template strand. This means that the sense strand or non-template strand of the parent double helix molecule has the same sequence as the mRNA (called the sense or coding strand). Dr Ian Mullaney Transcription – 3. Termination Elongation continues until Pol II hits an uninterrupted sequence of thymidine residues (polyT) on the template strand. This results in a series of transcribed of adenine residues (polyA tail) on the newly synthesised mRNA. This has two results. Termination of Pol II activity and final separation of DNA:mRNA helix formed during elongation. This new mRNA is termed the primary transcript and needs to be processed before translation can occur. https://www.youtube.com/ watch?v=SMtWvDbfHLo Dr Ian Mullaney Properties of the mRNA primary transcript The primary transcript can be characterised by the following. It contains: 1. A long sequence of adenine nucleotides (poly-A tail) on the 3’-end. 2. A cap on the 5’-end consisting of a molecule of 7methylguanosineattached “backward” (5’ to 5’) through a triphosphate linkage. 3. Untranslated sequences at either end of the coding region (the part that codes for a protein). 4. A coding region that contains the information to produce a mRNA is shortlived and is only produced protein. when needed then it is degraded. Thus at any time its cellular concentration is low. In fact, mRNA comprises only about 5% of the RNA in the cell but has the biggest size range (5006000 nucleotides). Dr Ian Mullaney Learning Objective 3 3. Be able to describe the basic steps that create lead to translatable mRNA in the cytoplasm. Dr Ian Mullaney Pretranslational processing of the mRNA primary transcript Where we currently are! Enhancer (distal control elements) Poly-A signal Termination sequence region Proximal control elements Exon Intron Exon Intron Exon DNA Upstream Promoter Chromatin changes Primary RNA transcript 5 (pre-mRNA) Exon Intron Transcription Intron RNA RNA processing mRNA degradation Coding segment Translation Protein processing and degradation Downstream Poly-A signal Exon Cleared 3end Intron Exon of primary transport RNA processing: Cap and tail added; introns excised and exons spliced together Transcription mRNA G P P P 5Cap 5UTR (untranslated region) Start codon Stop codon Dr Ian Mullaney 3UTR Poly-A (untranslated tail region) Pretranslational processing of the mRNA primary transcript The mRNA which has been transcribed up to this point is referred to as premRNA or primary transcript mRNA. Processing must occur to convert this into mature mRNA. This includes: Polyadenylation: The addition of a poly(A) tail to the 3′ end of mRNA. This stabilises RNA, which is necessary as RNA is much more unstable than DNA. This was covered during the discussion on the termination processes involved in transcription by Pol II. 5′ Capping: The addition of a methylated guanine cap to the 5′ end of mRNA. Its presence is vital for the recognition of the molecule by ribosomes and to protect the immature molecule from degradation by RNAases. Splicing: This allows the genetic sequence of a single preMRNA to code for many different proteins, conserving genetic material. This process is sequence dependent and occurs within the transcript. It involves: Dr Ian Mullaney 1. Removal of introns (non-coding sequences) Pretranslational processing of the mRNA primary transcript – 5’ Capping 5’ Capping: Capping is the first modification made to RNA polymerase II-transcribed RNA and takes place co-transcriptionally in the nucleus as soon as the first 25–30 nucleotides are incorporated into the recently formed transcript. In the eukaryotic cell, capping of mRNA 5′ ends is an essential structural modification that allows efficient mRNA translation, directs pre-mRNA splicing and mRNA export from the nucleus and limits mRNA degradation by cellular 5′–3′ exonucleases. 5’ capping, along with the poly(A) tail addition helps stabilise the mRNA molecule against cellular degradation. Capping enzymes attach an unusual nucleotide, 7methylguanosine, to the 5’ end of the pre mRNA. This results in a phosphate bridge attached to the C5 carbon of both sugars in the nucleotides. Dr Ian Mullaney Pretranslational processing of the mRNA primary transcript – RNA splicing RNA splicing is a process that removes the intervening, non-coding sequences of genes (introns) from premRNA and joins the coding sequences together in order to enable complete sequence translation of the mRNA into a protein. RNA splicing is mediated by the spliceosome. This is a large RNAprotein complex that catalyses the removal of the non-coding introns from pre-mRNA. The spliceosome comprises 3 major RNA-protein subunits termed small ribonucleoprotein particles Dr Ian (snRNPs) and an additional group ofMullaney Spliceosome assembly RNA splicing Pretranslational processing of the mRNA primary transcript – RNA splicing Thus RNA splicing removes non-coding regions from the pre-mRNA allowing for the exons to join together to form the mature finished transcript which can then be translated into the finished protein. This allows for one gene to ultimately produce one protein. However, the human genome project revealed that 30000 genes were able to code for 100000 proteins. This is achieved using alternative splicing whereby one gene can produce Dr Ian Mullaney numerous, distinct protein products. RNA splicing Alternative RNA splicing Pretranslational processing of the mRNA primary transcript – RNA splicing Tropomyosins are rodlike dimeric proteins which form head to tail polymers along the length of actin filaments. There are numerous forms of the protein depending on cell type, all arising from a single alternatively spliced gene. Dr Ian Mullaney Pretranslational processing of the mRNA primary transcript – Nuclear export RNA splicing results in the production of the mature mRNA which is ready to leave the nucleus and enter the cytoplasm of the cell, the site of protein translation. The cell nucleus contains pores in the nuclear membrane allowing passage Dr Ian Mullaney Learning Objective 4 4. Recognise that aberrant gene expression is a major cause of disease. Dr Ian Mullaney Aberrant gene expression and RNA processing as an indicator of disease 1. Aberrant gene expression General transcription factors and chromatin regulators: • fibroids and prostate cancer, as well as developmental disorders • Loss of function of nucleosome remodellers in a number of cancers Transcription factors • cMyc-one most frequently amplified oncogene • AIRE-gene defects lead to autoimmune disorders • HNF1a/b, HNF4a-maturity onset diabetes of the young Dr Ian Mullaney Aberrant gene expression and RNA processing as an indicator of disease 2. Aberrant RNA splicing Mutated splice sites: • cancer (BRCA1 and 2) • spinal muscular atrophy (SMN2) • Frasier syndrome (WT1)- kidney disease and males ambiguous genitalia • atypical cystic fibrosis (CFTR) Mutated splicing machinery: • retinitis pigmentosa (spliceosome components) • spinal muscular atrophy (snRNPassembly -small nuclear ribonucleotides) • myotonic dystrophy and glioblastoma (alternative splicing Dr Ian Mullaney factors) Transcription – An overview Enhancer (distal control elements) Poly-A signal sequence Proximal control elements Exon Intron Intron Exon Termination region Exon DNA Downstream Upstream Promoter Chromatin changes Transcription Exon Primary RNA 5 transcript (pre-mRNA) Intron Exon Poly-A signal Intron Exon Cleared 3end of primary transport RNA processing: Cap and tail added; introns excised and exons spliced together Transcription Intron RNA RNA processing mRNA degradation Coding segment Translation Protein processing and degradation mRNA G P P P 5Cap 5UTR (untranslated region) Dr Ian Mullaney Start codon Stop codon 3UTR (untranslated region) Poly-A tail Diagrams adapted from: Essential Cell Biology, Alberts et al, 6th ed. Molecular Biology of the Cell, Alberts et al, 4th ed. Molecular Cell Biology, Lodish et al, 4th ed. Acknowledgement to Dr Nandini Hayes, Institute of Health Sciences Education