Many mRNA transcripts and mature noncoding RNAs have nucleotides at their ends that were not directed by transcription of the sense strand of the respective gene. Instead, the specific end nucleotides or oligonucleotides were covalently attached by processing enzymes. The effect is known, or thought, to be to enhance the stability of the RNA or assist it in carrying out an important function.
In addition to RNA splicing, the ends of RNA polymerase II transcripts undergo modifications. The 5′ end is capped by adding a specific variant guanine that is added by means of an unusual and distinctive type of phosphodiester bond, and a long sequence of adenines is added to the 3′ end. As well as protecting the ends of the RNA from cellular exonucleases, these modifications may assist the correct functioning of the RNA transcripts. The addition of the trinucleotide CCA to the 3′ end of transfer RNAs is important for tRNA function.
5′ Capping
Shortly after transcriptional initiation by RNA polymerase II, a methylated nucleoside (7-methylguanosine, m7G) is added and linked by a 5′–5′ phosphodiester bond to the first 5′ nucleotide. This is a major feature of an end-addition form of RNA processing known as capping of the 5′ end of the transcript (Figure 1). 5′ Capping is thought to serve diverse functions, including: • Protecting the transcript from 5′ → 3′ exonuclease attack (the uncapped RNA transcripts are rapidly degraded);
• Facilitating transport of mRNAs from the nucleus to the cytoplasm;
• Facilitating RNA splicing; and
• Facilitating attachment of the 40S subunit of cytoplasmic ribosomes to mRNA during translation.
Fig1. The 5′ cap of a eukaryotic mRNA. The 5′ end of a eukaryotic mRNA has a specialized cap that provides protection against exonucleases and has various other functions, including assisting initiation of translation (see text). The capping process involves: (i) removal of the gamma (γ) phosphate of the original terminal 5′ nucleotide, which is normally a purine (Pu); (ii) addition of a GMP (derived from a GTP precursor) through a 5′-5′ triphosphate linkage (gray shading); (iii) methylation of nitrogen atom 7 of the new 5′ terminal G to produce 7-methylguanosine (m7G). In mRNAs synthesized in vertebrate cells, the 2′ carbon atom of the ribose of each of the two adjacent nucleotides is also methylated, as illustrated by pink shading. N, any nucleotide.
3′ Polyadenylation
Transcription by both RNA polymerase I and III stops after the enzyme recognizes a specific transcription termination site. However, the 3′ ends of mRNA molecules are determined by a post-transcriptional cleavage reaction. As RNA polymerase II advances to transcribe a gene, it carries at its rear two multiprotein complexes, CPSF (cleavage and polyadenylation specificity factor) and CStF (cleavage and stimulation factor), that co-operate to identify a specific hexanucleotide polyadenylation signal, often AAUAAA, located downstream of the termination codon in the RNA transcript. Thereafter, the RNA is cleaved at a specific site 15–30 nucleotides downstream of the AAUAAA sequence (although the primary transcript may continue for hundreds or even thousands of nucleotides past the cleavage point).
After cleavage has occurred, in mammalian cells about 200 adenylate (AMP) residues are added sequentially by the enzyme poly(A) polymerase. This polyadenylation reaction (Figure 2) produces a poly(A) tail that is thought to:
• Help transport mRNA to the cytoplasm;
• Stabilize at least some mRNA molecules in the cytoplasm; and
• Enhance recognition of mRNA by the ribosomal machinery.
Fig2. Polyadenylation of 3′ ends of eukaryotic mRNAs. (A and B) RNA polymerase II transcribes the template strand of a gene, and as it does this it carries at its rear multiprotein complexes including two required for polyadenylation: CPSF (cleavage and polyadenylation specificity factor) and CStF (cleavage and stimulation factor). They co-operate to identify a polyadenylation signal downstream of the termination codon in the RNA transcript and to cut the transcript at the point marked by the yellow dart. The polyadenylation signal comprises an AAUAAA sequence or close variant and some poorly understood downstream signals. (C) Cleavage occurs normally about 15–30 nucleotides downstream of the AAUAAA element and (D) AMP residues are subsequently added by poly(A) polymerase to form a poly(A) tail.
3′ CCA addition to tRNAs
All mature transfer RNAs have at their 3′ terminus the sequence CCA, but in eukaryotes this sequence is not copied from the sense strand of the tRNA gene. Instead, it is added by a nucleotidyltransferase in the nucleus. This sequence is vital for the function of a tRNA and ensures correct recognition of the mature tRNA by an enzyme that will covalently link the correct amino acid to the end adenosine. Any tRNA that has not been correctly processed in this way will not be permitted export to the cytoplasm.
