Hallbrook Lec 3-RNA processing

• eukaryotic mRNAs uniquely undergo several key steps: 1)addition of 5' cap, 2)addition of poly(A) tail, 3)splicing, 4)nuclear export
• pre-mRNA is the immediate production of transcription while mRNA is the fully processed transcript
• every transcript gets a modification made to its 5' end shortly after transcription begins which is the 5' cap (only poly II transcripts are capped)
• the 5' cap 1)protects the 5' end of the transcript from degradation by 5' to 3' exonucleases, 2)has a molecular handle used by many proteins that bind to transcripts, 3)required for traditional translation
• the capping molecule of a 5' cap is 7-methylguanosine which is a GTP that is added backwards onto the 5' phosphate of the transcript and then methylated at the 7 position (sometimes the first 2 bases of the mRNA gets methylated too)
• the 5' cap is added via an enzyme called guanylyltransferase aka the "capping enzyme"
• guanylyltransferase binds to the CTD of poly II and as the mRNA leaves poly II, the 5' end sticks to the CTD which allows guanylyltransferase to cap it
• after the 5' cap is added, the guanylyltransferase falls off and a protein called CBC(cap-binding complex) binds to the cap, and the CBC at the 5' end of the transcript remains tethered to the CTD
• mRNA capping also helps recruit the transcript to the ribosome for subsequent translation
• to show that mRNA capping is a necessary process to recruit mRNA to the ribosome, we use a combination of radio-labels and density-gradient centrifugation
• capped mRNA is mixed with ribosomes to allow binding, is ran through a density gradient so ribosomes sink to the bottom, and if mRNA binds to ribosomes it will sink to the bottom as well
• capped mRNA binds to ribosomes and allows detection where capped mRNA ended up by following 3H signal
• polyadenylation is a long tail of adenosines (80-250) added to the 3' end of every mRNA and is called a poly(A) tail
• a poly A tail protects the 3' end from degradation and helps recruit to ribosomes for translation
• the 1st step to adding a poly A tail is that the poly II transcribes past the termination sequence or poly A addition site(AAUAAA) where the cleavage-poly adenylation cleavage factors(CPSF) attached to the CTD cuts the mRNA downstream of the AAUAAA before GU rich region
• step 2 of adding a poly A tail is where polyadenylate polymerase(PAP) begins adding adenosines and the poly A binding protein (PABP) attaches to the tail, protecting it
• eukaryotic mRNA transcripts initially have a lot of extra sequence that doesn't encode amino acids and these have to be removed before the mRNA can be translated via splicing
• exons are parts of the mRNA sequence that are present in the final transcript - expresses protein
• introns are regions in between exons that don't encode amino acids and are removed by splicing - interrupts coding sequence
• most exons encode amino acids except: 1)5' UTR which contains sequences that help initiate translation, and 2)3' UTR that contains the poly A addition site and some regulatory sequences
• introns contain sequences involved in gene regulation (ie. intronic enhancers)
• alternative splicing occurs when more than one type of mRNA can be produced from a single gene so some exons can be entirely or partially skipped
• alternatively spliced mRNAs are also called isoforms
• in alternative splicing, exons are NOT re-arranged in splicing and remain in numerical order
• most genes (~90%) have multiple isoforms
• in addition to alternative splicing of introns, a gene can have more than one site for cleavage and poly A attachment
• one gene can produce multiple protein products via alternative splicing AND poly A site choice
• the splicing machinery knows where the introns and exons are by sequences in the intron
• all introns contain: 1)5' splice site - typically GU, plus other sequence, 2)3' splice site - typically AG, plus other sequence, 3)branch site - internal A nucleotide, just upstream of 3' splice site

  
• aside from GU and AG, exact sequences of 5' and 3' splice sites are difficult to define
• the 1st step of the splicing mech is that the branch 2' OH attacks 5' splice site and severs the bond between exon and intron at 5' site instead attaching the branch point to 5' splice site on intron
• the 2nd step in splicing mech is that the severed 5' exon is "activated" to attack the 3' splice site, 3' OH of 5' exon is free to attack 3' splice site, severs bond btwn exon and intron at 3' site, and attaches 5' exon to 3' exon
• the 3rd step in splicing mech is when the intron is released as "lariat" shape

  
• spliceosomes are the machinery that does the splicing mechanism
• snRNPs are small nuclear ribonucleoproteins - complexes that form the spliceosome
• snRNAs are small nuclear RNAs, the heart of the snRNPs, ~100-200 nucleotides
• 5 snRNPs in the spliceosome are: U1, U2, U4, U5, and U6
• U1 is both a snRNP and a snRNA
• snRNPs facilitate each step of the splicing reaction
• snRNPs are recruited essentially in numerical order