BIOL 2960 Lecture 33: (33-35): RNA Processing

10 Pages
Unlock Document

Biology And Biomedical Sciences
Biology And Biomedical Sciences BIOL 2960
Kunkel Barbara

Lecture 33-35 Sunday, April 23, 2017 5:59 PM RNA PROCESSING Remember: RNA is read in the 5' --> 3' direction, so the complementary sequence should be reported in the 3' --> 5' direction RNA procession in eukaryotes: • 5' mRNA capping • Cleaving and poly(A) addition to generate the 3' end of the mRNA • Splicing = removal of intron sequence • Need to know how to draw this: • mRNA processing occurs in the nucleus, prior to export to the cytoplasm • Capping ○ Mechanism ▪ Steps: □ Start off with tri-phosphate tail □ One phosphate is removed □ GTP loses two phosphates and then attaches itself to the two at the tail □ Add methyl group to base □ Add methyl group to ribose (only sometimes) ▪ Note: cap addition occurs shortly after transcription is initiated -- occurs as it is still being transcribed. Coordinated by the phosphorylated PolII CTD (C-terminal domain) -- enzymes are associated with the CTTD tail that facilitate these processes ○ Coupling to Transcription: in our cells, 5' CAP addition, an early step in transcription, is coupled to initiation 1) TFIIH kinase phosphorylates CTD on Ser5 of sequence 2) A "stall factor" stops PolII shortly downstream of initiation site 3) CAP addition complex binds to Ser5-phosphorylated CTD of stalled polymerase, and caps 5' end of mRNA 4) A second kinase phosphorylates the stall factor to release the polymerase and also phosphorylates the CTD on Ser2 5) Additional factors are recruited to Ser2-phosphorylated CTD. These facilitate splicing and passage of polymerase through downstream chromatin. ○ Functions of the 5' cap: ▪ Increase transcript stability by protecting against ribonucleases (enzymes that break down RNA) ▪ Increase translation efficiency -- 5' cap binds to translation initiation factors -- equivalent of the Shine-Delgarno sequence for eukaryotes • Polyadenylation ○ Definition: addition of a poly(A) tail to the 3' end of pre-mRNA ○ Proteins carried by polymerase responsible for polyadenylation search for a cleavage signal, often AAUAAA -- approx. 20 bases downstream from the signal sequence, the polyadenylation machinery will make a cut and add the A's ○ • Intron Splicing ○ Exon/intron distribution ▪ Introns are large; exons are small; splicing is accurate! ▪ Most of the exons in human genome are between 0 and 300 nucleotides long -- quite short, because introns tend to be between 100-2000 nucleotides long! ○ Mechanism ▪ Signals □ They just kind of squeeze together such that the intron is excised in the form of a "lariat" □ □ Conserved consensus sequences (sequence of DNA similar in structure in different organisms) exist at the junction between introns and exons that are therefore related to intron splicing ▪ Chemistry □ There are two steps in splicing; both are classified as trans-esterification reactions, where a phosphodiester bond is broken and remade □ Need to memorize this:  2'OH of the A at the branch point attacks the P at the 5' splice site (3' end of the exon) between the conserved GU and exon  This generates the lariat intermediate and frees the 3'OH end of the G on the exon  This 3'OH of the exon attacks the phosphate between the intron and the downstream exon at the conserved GA sequence (3' splice site, which is the 5' end of the second exon)  This releases the lariat and results in the spliced product!   Note: the bond formed for the lariat intermediate is a 2'-5' phosphodiester linkage, so it's a little different than usual phosphodiester linkage, so it's a little different than usual  □ What enzymes are involved:  Multiple RNA/protein complexes called snRNPs (small nuclear ribo- nucleoproteins:  U1, U2, U4, U5, U6 ▪ snRNPs: small nuclear ribonucleoproteins □ U1, U2, U4, U5, U6 □ Role of the snRNPs  U1: Binds the 5' splice site  U2: Binds the branch site and forms part of the catalytic center  U5: Binds the 5' splice site and then the 3' splice site  U4: Masks the catalytic activity of U6  U6: Catalyzes splicing □ Step 1: Splicing begins with recognition of the 5' splice site by the 165 base long U1 snRNA found in the U1 snRNP □ snRNA recognizes the consensus sequence at the 5' splice site via the principle of complementary anti-parallel RNA □ □ Step 2: Splicing continues with the binding of a protein complex (PC) to the 3' splice site, which then recruits the U2 snRNP to the Branch Point □ Step 3: U4-U5-U6 complex comes in and all of the snRNPs together form the spliceosome  U5 brings the two exons into close proximity  U4 and U6 are bound together -- U4 is the chaperone (keeps U6 inactive until it reaches the spliceosome) and U6 is the heart of the catalytic site □ Step 4: U1 and U4 are released, leaving U2, U5, U6 for catalysis, which is an RNA/protein complex  U4 lets go of U6, and when U1 lets go, U6 takes its place and binds to the 5' splice site  U6 and U2 become closely associated □ Step 5: First transesterification and lariat formation □ Step 6: Second transesterification, yielding spliced exons and lariat intron □ Electron micrograph: □ An RNA-centric view of splicing shows that it is directed and probably catalyzed by the small nuclear snRNP RNAs by the small nuclear snRNP RNAs □ Note: the branch point does not pair with the U2 sequence, it is excluded and "presented" such that it can attack the phosphodiester bond at the 5' splice site □ This whole unit is an RNA enzyme responsible for the transesterification reactions □ Note: the molecular mechanism of splicing is super complex and is still being actively investigated!! ○ Regulation: how cells define exons ▪ Alternative splicing patterns □ Alternative
More Less

Related notes for Biology And Biomedical Sciences BIOL 2960

Log In


Join OneClass

Access over 10 million pages of study
documents for 1.3 million courses.

Sign up

Join to view


By registering, I agree to the Terms and Privacy Policies
Already have an account?
Just a few more details

So we can recommend you notes for your school.

Reset Password

Please enter below the email address you registered with and we will send you a link to reset your password.

Add your courses

Get notes from the top students in your class.