Question

Eukaryotic mRNA has both a 5’ cap and a 3’ poly(A) tail.How are they produced?

Answer 1

Eukaryotic mRNA is required to be processed before transporting it out of the nucleus for translation. There are 3 major events- capping of the 5’ end of the mRNA, mRNA splicing and polyadenylation of its tail. RNA splicing is the process where the introns are removed from the mature mRNA. However, this question has asked about the other two mechanisms, that is, 5’ capping and 3’ polyadenylation of the mRNA.

5’ Capping

1. RNA processing is a simultaneous activity along with transcription. Infact, mRNA is capped just the moment it emits out from the exit channel when RNA polymerase shifts from initiation to elongation phase of transcription. One protein hSPT5 (an elongation factor) recruits the 5’ capping enzyme to the phosphorylated serine (position 5) in the carboxy terminal domain of RNA polymerase. RNA polymerase (II) is the enzyme required for eukaryotic mRNA transcription.

2. Capping is the process where the 5’ end of the mRNA is attached to a methylated guanine residue by a 5’-5’ linkage. 5’ cap is produced by three enzymatic steps- first a phosphate is removed from the 5’ end of the mRNA, second a GMP is added, lastly the GMP is modified by addition of a methyl group.

3. Finally, after 5’ capping, the serine at position 5 in the carboxy terminal domain of RNA Polymerase II is dephosphorylated to remove the capping machinery.

3’ Polyadenylation of the tail

1. Polyadenylation is linked with termination of transcription. The carboxy terminal domain of the RNA polymerase enzyme is involved in recruiting some of the enzymes required for the event. Once RNA polymerase reaches the end of the gene, it encounters some sequences which trigger loading of enzymes required for 3’ polyadenylation.

2. Two proteins are carried by carboxy terminal domain of RNA polymerase, CPSF (cleavage and polyadenylation specificity factor) and CstF (cleavage stimulation factor). As RNA polymerase transcribes the end of the gene, proteins CPSF and CstF are loaded onto the mRNA (poly A signals) resulting into RNA cleavage and polyadenylation.

3. Polyadenylation is catalyzed by an enzyme called poly A polymerase which adds approximately 200 adenine nucleotides to the 3’ tail of the mRNA. The enzyme uses ATP as precursor and elongates the poly A chain like RNA polymerase elongates the mRNA. It is not well understood what determines the length of the poly A tail in eukaryotic mRNA. However, this feature is exclusive for eukaryotic protein coding mRNAs transcribed by RNA Polymerase II.

4. Finally, mature mRNA can be transported out of the nucleus. But what about the RNA polymerase enzyme that is still adhered to the template DNA strand. It continues to transcribe a second RNA chain which when emits out through the exit channel is not capped and degraded rapidly. Human RNAse Xrn2 is loaded onto the end of the new RNA by another protein Rtt103. Xrn2 is highly processive, it continues to degrade the new RNA chain in a 5’->3’ direction until it reaches the RNA polymerase enzyme. Torpedo model suggests that Xrn2 either pushes the polymerase forward or it might pulls out the entire new RNA chain and terminates RNA transcription finally. Another alternative model, known as allosteric model suggests that RNA polymerase undergoes conformational change and loses its processivity, so it falls off from the DNA template strand.