RNA Processing Splicing and the Spliceosome PDF

RNA Processing Splicing and the Spliceosome BMS 532 BLOCK 3 L EC TURE 6 (OV ERA LL LEC T URE 21)  Objectives 1. Define the following terms or processes: deamination, colinear vs split, polyadenylation, methyltransferase, exon, intron, lariat, snRNA, snRNP, spliceosome, upstream eleme...

RNA Processing Splicing and the Spliceosome BMS 532 BLOCK 3 L EC TURE 6 (OV ERA LL LEC T URE 21)  Objectives 1. Define the following terms or processes: deamination, colinear vs split, polyadenylation, methyltransferase, exon, intron, lariat, snRNA, snRNP, spliceosome, upstream elements, downstream elements, and regulatory elements 2. Identify the consequence of base deamination and Explain the role of RNA modification/RNA Editing in generating unique protein products 3. Compare and contrast RNA processing and editing in prokaryotes vs. eukaryotes 4. Describe the basic structure of RNA molecules with emphasis on processed Eukaryotic mRNA and the components added to generate the final version exported from the nucleus 5. Explain the role of mRNA processing in eukaryotes and list the stages and factors involved in mRNA processing ◦ Explain the importance of mRNA capping and the role of methyltransferases in mRNA capping 6. Compare and contrast snRNA and mRNA in terms of gene structure, elements located within the DNA, and processing 7. Explain the role of snRNPs in the spliceosome and identify the role in splicing for each of the following: U1, U2, U4, U5, and U6 8. Summarize the stages of major spliceosome assembly and splicing in eukaryotes with emphasis on the activity of the snRNPs, helicases (Prp5, Sub2, and Prp22), Complexes (A, B, and C), and post-spliceosomal complex and explain how recent advances have changed the understanding of vertebrate spliceosome assembly and maturation 9. Summarize and explain alternative splicing, its regulation, and its role in increasing functional diversity in biological systems  Modification of RNA: RNA Editing RNA Editing = making changes in the sequence of an RNA molecule Additions or deletions of bases can occur to change the sequence of an RNA molecule Bases can also be converted from one into another ◦ Deamination is a major mechanism for these changes ◦ NOTE: Provides a natural, desired process for the base modifications discussed in the mutations section These edits can change the final protein product and enable critical variation for function ◦ EXAMPLE ◦ Apolipoprotein B-100 unedited form = longer ◦ Apolipoprotein B-100 deamination of a single cytosine creates a premature stop codon and a truncated protein molecule ◦ Thus, 2 different functional forms of the same protein can be created from a single gene LO2  Introduction to RNA Processing Processing of RNA molecules is critical to function and stability Takes a variety of forms including association with RNA binding factors or induction of cleavage events Most RNA molecules have been observed as being processed in some way with underlying physiological need Processing can be regulatory in nature to ensure activities only take place when required LO3  Transcription and Processing in Prokaryotes and Archaea Transcription and Translation in prokaryotes is coupled as they are not separated in physical space The processing of the molecules is therefore different and involves colinear coding (all bases in a sequence are present in tandem) RNA sequences can still undergo complex processing that assists with proper function Processing for these organisms often involves cleavage events with intron-like portions being removed LO3 Clouet-d’Orval et al 2018  Eukaryotic mRNA In many eukaryotic genes the coding regions which specify the structure of proteins are interrupted by noncoding segments = “split genes” Coding regions = exons Noncoding regions = introns Primary transcript contains exons and introns; introns are subsequently removed = “splicing” There is no such thing as “junk” DNA!! LO3, LO4, LO6  Eukaryotic Transcription: Processing The first processing step adds 7- methylguanosine to 5’ end = “cap” Additional processing involves polyadenylation (the addition of a series of adenines at the 3’ end of the transcript) = “poly A tail” The processed transcript contains the 5’ cap, adjacent exons, and a poly A tail Processing plays a role in the half-life of the RNA molecule LO3, LO4, LO5, LO6  mRNA Capping via Methyltransferases Forms on the first nucleotides of RNA transcribed by RNA pol II Variable structure across organisms ◦ In mammals main form = m7G(5′)ppp(5′)Xm ◦ 7-methylguanosine (m7G) is linked to the first transcribed nucleotide (X) via a 5′ to 5′ triphosphate bridge CAPAM is a capping methyltransferase that specifically caps adenosine nucleotides in the first position (Xm) Protect mRNA from nucleases and innate immune responses LO5 Cowling 2019  Transcription and Processing: snRNA vs. mRNA Small nuclear RNA (snRNA) and mRNA both undergo processing Arrangement of upstream and downstream elements within the DNA of these genes is very similar and essential to transcription DSE = distal sequence element ◦ Acts like an enhancer PSE = proximal sequence element ◦ Acts like a promoter Differences ◦ snRNA ◦ LEC = little elongation complex ◦ GTFs = general transcription factors ◦ SNAPc = snRNA-activating protein complex ◦ mRNA ◦ SEC = super elongation complex ◦ Splicing factors and use of spliceosome LO6 Matera and Wang 2014  snRNA and Processing snRNA are essential to the function of the spliceosome Additional processing to generate functional snRNA occurs Both cytoplasmic and nuclear regulatory steps are involved ◦ Export and then Import LO6 Matera and Wang 2014 Splicing and the Spliceosome DETAILED DISCUSSION OF A MAJOR COMPONENT OF RNA PROCESSING  RNA Processing: Splicing and the Spliceosome Spliceosomes contain protein and specialized small nuclear RNAs (snRNA) complementary to the splice junctions to provide specificity to splicing reaction ◦ NOTE: Although there is a minor spliceosome, we will focus on the major spliceosome mechanism as the two are similar in nature snRNAs combine with protein to form snRNPs snRNPs U1, U2 and U5 recognize splice donor and acceptor sites by complementary base pairing so that intron excision is precise This process will be discussed in more detail with spliceosome assembly LO7, LO8  RNA Transcription: Splicing After recognition, the 5’ exon moves to the 3’ splice acceptor site where a second cut is made by the spliceosome Exon termini are joined and sealed The loop is released as a lariat structure which is degraded The spliced mRNA contains fused exons with coding information only Alternative splicing increases variety in products LO7, LO8  Spliceosome Assembly and Activity Spliceosome assembly occurs at sites of transcription ◦ Begins across exon with rearrangement occurring to span neighboring intron snRNA can associate with the pre-processed transcript of mRNA in a manner mediated by C- terminal domain of pol II ◦ Forms pre-spliceosome complex A ◦ Requires use of helicases Prp5 and Sub2 The initiation and recognition steps are essential to ensure introns are removed and the desired exons spliced together (splicing can be alternative and variable for some mRNA molecules) Component snRNPs are released and recycled for future use at the end of the process LO7, LO8 Matera and Wang 2014  Spliceosome Assembly and Activity: Full Process Starts along with Transcription @ site of active transcription U1 and U2 snRNPs associate via recognition of 5’ and 3’ splice sites ◦ Their interaction = Complex A (a.k.a pre-spliceosome) This complex is joined by a complex of 3 additional snRNPs (U4/U6 and U5) which together with Complex A = Complex B (a.k.a. the pre-catalytic spliceosome = catalytically active) ◦ Complex B requires multiple RNA helicases ◦ Helicase activity results in release of U4 and U1 (lariat precursor is formed) and ◦ Complex C is generated after Complex B carries out first catalytic step Complex C undergoes rearrangement and carries out second catalytic step ◦ Forms post-spliceosomal complex (involves combination of complex with lariat intron and spliced exons) snRNPs U2, U5, and U6 are released from particle ◦ Lariat intron released and exons are spliced ◦ Release of spliced particle (i.e. mRNA) via helicase Prp22 LO7, LO8 Matera and Wang 2014  Additional Complexity of Complex B In general for humans and other vertebrates, exons are much smaller than introns A conversion from Exon Defined (ED) spliceosomes to Intron Defined (ID) spliceosomes occurs prior to the activation of the catalytic spliceosome ◦ Formation at exon then rearrangement to intron Cryo-electron microscopy identified 4 unique states for human ED spliceosomes with implications for the processing of human mRNA (2024 update) ◦ Implies a maturation process for spliceosomes: ◦ The pre-B to B complex formation is actually a series of regulated mechanistic steps ◦ Factors are recruited in a step-wise fashion with specific recognition of the exon 3’ region ◦ Provides insights into canonical splicing, back-splicing, and exon skipping (a component of alternate splicing) LO8 Zhang 2024  Alternative Splicing A system limited to only a single gene product from a sequence would not need to separate the DNA sequence as is observed in eukaryotes The ability to generate multiple similar products from the same sequence is critical to increasing options for system function Having introns and exons enables multiple products to be formed from the same sequence ◦ Same regulatory control mechanisms for transcription ◦ New element of regulatory or function control at the level of transcript processing LO9 Guttmacher and Collins 2002  Regulation of Alternative Splicing Choosing the splice site is regulated via a combination of cis-regulatory elements and trans-acting factors ◦ Cis-acting regulatory elements known as Splicing Regulatory Elements (SREs) ◦ SREs have several designations (ESE, ISE, ESS, or ISS) and can be located in exons or introns ◦ Can promote (enhancer) or prevent (silencer) splicing ◦ Trans-acting splicing factors ◦ Recruited to the site by the SREs Activity of the factors depends on the situation with multiple activities possible on the same transcript LO9 Matera and Wang 2014 Questions What is the difference between prokaryotic gene sequences and eukaryotic gene sequences? (LO3) In what ways are the structures of snRNA genes similar to mRNA genes? (LO6) What are the roles for the various snRNPs in splicing? (LO7,LO8) What regulatory element has the potential to control the final form of the mRNA? (LO9)