M3 Mosher- mRNA splicing and processing

how is co-transcriptional mRNA processing coordinated?

• RNA pol II has a CTD ‘tail’ with many copies of a heptad amino acid repeat

• this is initially unphosphorylated

• differential phosphorylation (particularly at serine 2s and 5s) of this CTD during transcription recruits the machinery for capping, splicing and polyadenylation at different points

• this mediates initiation, elongation and termination

how is eukaryotic transcription initiated? what regulatory steps can control this?

• TATA-binding protein (TBP) recognises and binds the TATA box upstream and recruits general transcription factors (GTFs) to assemble the pre-initiation complex (PIC) at the transcription start site

• RNA Pol II melts the DNA to form the transcription bubble

• polymerisation begins without a primer, but Pol II is still bound to the GTFs, causing DNA scrunching

• the GTFs should release from the DNA and Pol II, but sometimes they are bound too tightly and polymerase falls off instead and has to try again, in abortive transcription

even if Pol-II can continue, other signals can cause promoter-proximal pausing of Pol-II, which halts the process, either to:

• sterically block additional transcription/trigger premature termination

• act as another regulatory step of ‘poising’, so the gene can be transcribed immediately following other signals

• ensure that the 5’ cap is assembled correctly (quality control)

what could proximal causing of transcription be used for?

promoter-proximal pausing of Pol-II after initiation halts transcription, either to:

• sterically block additional transcription/trigger premature termination

• act as another regulatory step of ‘poising’, so the gene can be transcribed immediately following other signals

• ensure that the 5’ cap is assembled correctly (quality control)

how are 5’ caps placed co-transcriptionally?

• the 5’ cap helps indicate that an mRNA is intact

• the proteins that place a 5’ cap are recruited co-transcriptionally by phosphorylation signals (serine 5 but not serine 2) in the CTD of RNA Pol II

• the 5’ triphosphate RNA nucleotide is dephosphorylated

• guanosine monophosphate is added ‘upside down’, with a 5’ to 5’ linkage to the diphosphate nucleotide

• methylation of nucleotides

how are 3’ polyA tails placed co-transcriptionally?

• the polyA tail helps indicate that an mRNA is intact

• the proteins that place a polyA tail are recruited co-transcriptionally by phosphorylation signals (serine 2 but not serine 5) in the CTD of RNA Pol II

• the 5’ triphosphate RNA nucleotide is dephosphorylated

• guanosine monophosphate is added ‘upside down’, with a 5’ to 5’ linkage to the diphosphate nucleotide

•