Lecture 1 - RNA Cap Synthesis and Function

Question 1

RNA Cap:

Answer 1

7-methylguanosine linked by triphosphate to the first transcribed nucleotide

mRNA cap flags mRNA for specific processing events (e.g. splicing, export, translation) and prevents other RNAs from being processed in the mRNA pathway

Question 2

Functions of RNA Cap:

Answer 2

Protects RNA from degradation:
-Without the RNA cap, triphosphate at the 5’ end of RNA is susceptible to attack from exonucleases

RNA cap forms a recruitment surface for proteins and complexes involved in RNA processing:

• CBC (Cap Binding Complex):
• • Involved in splicing, mRNA export
• • 2 subunits
• • Binds RNA cap early in transcription
• eIF4F (eukaryotic Initiation Factor 4F):
• • Involved predominantly in translation initiation
• • Binds later than CBC

Question 3

How does the RNA cap form only on RNA Pol II transcribed RNA?

Answer 3

RNA Pol II makes pre-mRNA

RNA Pol II has a special domain called the C-terminal domain (CTD) which recruits the enzymes which make the cap.

RNA Pol II’s CTD is phosphorylated at the initiation of transcription by TFIIH, a basal transcription factor.

The CTD has 52 repeats of almost the same amino acid sequence “YSPTSPS”.

TFIIH phosphorylates the CTD at Ser5

This recruits and activates the first capping enzyme (CE aka RNGTT) and later recruits the second capping enzyme (RNMT).

Question 4

RNA Cap Formation:

Answer 4

2 Step Process:
Step 1: Guanosine Capping
Step 2: mRNA cap methylation

Step 1:

• Involves CE (Capping Enzyme) which has triphosphatase and guanylyl transferase activities
• CE uses GTP to make the cap
  1. RNA made with 5’ triphosphate
  2. CE hydrolyses 1st phosphate to make 5’ phosphate
  3. CE binds GTP
  4. CE hydrolyses GTP and binds GMP
  5. GMP is joined to ppX-RNA to make basic cap (X = 1st nucelotide)

Step 2:

• Involves RNMT which is a cap methyltransferase
• RNMT uses s-adenosyl methionine (SAM) as a methyl donor
  1. Basic cap from step 1 + SAM -> 7-methylguanosine cap

NB: Other cap methyltransferases exist: e.g. CMTR1, CMTR2, CAPAM