Eukaryotic mature mRNA structure

Background

Chemical structure of RNA

For further information, see RNA and Messenger RNA

Messenger RNA (mRNA) is RNA that has a coding region that acts as a template for protein synthesis (translation). The rest of the mRNA, the untranslated regions, tune how active the mRNA is. The are also many RNAs that are not translated, called non-coding RNAs. Like the untranslated regions, many of these non-coding RNAs have regulatory roles.

The name poly(A) tail (for polyadenylate tail) reflects the way RNA nucleotides are abbreviated, with a letter for the base the nucleotide contains (A for adenine, C for cytosine, G for guanine and U for uracil). Nucleotides are the individual units that make up RNA. RNA sequences are written in a 5' to 3' direction, where the 5' end is the part of the RNA molecule that is transcribed first, and the 3' end is the last transcribed part (transcription is the synthesis of RNA from a DNA template). The 3' end is also where the poly(A) tail is found, if it is a polyadenylated RNA.

Stabilising polyadenylation

Function

The poly(A) tail protects the mRNA molecule from degradation by exonucleases in the cytoplasm and aids in transcription termination, export of the mRNA from the nucleus, and translation. Almost all eukaryotic mRNAs are polyadenylated.^[1][2]

Many eukaryotic non-coding RNAs are always polyadenylated at the end of transcription. There are small RNAs where the poly(A) tail is only seen in intermidiary forms and notin the mature RNA as the ends are removed during processing. MicroRNAs are such a case. For large non-coding RNAs, which are processed similarly to mRNAs, the poly(A) tail is part of the final RNA. Xist, which mediates X chromosome inactivation, is such an RNA.^[3][4]

Mechanism

The polyadenylation machinery in the nucleus of eukaryotes works on products of RNA polymerase II, such as precursor mRNA. Here, a multi-protein complex (see components on the right) cleaves the 3'-most part of a newly produced RNA and polyadenylates the end produced by this cleavage. The cleavage is catalysed by the enzyme CPSF^[5] and occurs 15?30 nucleotides downstream of its binding site.^[6] This site is often the sequence AAUAAA on the RNA, but variants of it exist that bind more weakly to CPSF.^[7] Two other proteins add specificy to the binding to an RNA: CstF and CFI. CstF binds to a GU-rich region further downstream of CPSF's site.^[8] CFI recognises a third site on the RNA (a set of UGUAA sequences in mammals) and can recruit CPSF even if the AAUAAA sequence is missing.^[9][10] The RNA is cleaved right after transcription, as CstF also binds to RNA polymerase II.^[11] The protein CFII is also involved in cleavage somehow.^[12] When the RNA is cleaved, polyadenylation starts, catalysed by polyadenylate polymerase. Polyadenylate polymerase builds the poly(A) tail by adding adenosine monophosphate units from adenosine triphosphate to the RNA, cleaving off pyrophosphate.^[13] Another protein, PABPN1, binds to the new, short poly(A) tail and increases the affinity of polyadenylate polymerase for the RNA. When the poly(A) tail is approximately 250 nucleotides long the enzyme can no long bind to CPSF and polyadenylation stops, thus determing the length of the poly(A) tail.^[14][15] CPSF is in contact with RNA polymerase II, and promotes it to terminate transcription.^[16] The polyadenylation machinery is also linked by CFI to the spliceosome, a complex that removes introns from RNAs.^[10]

The polyadenylation signal ? the sequence motif recognised by the RNA-binding factors CFI, CPSF and CstF ? varies from between groups of eukaryotes. A majority of human polyadenylation sites contain the AAUAAA sequence,^[8] but it is less common in plants and fungi.^[17]

Downstream effects

A fragment of a poly(A) binding protein binding to a poly(A) sequence

Poly(A) binding protein also stimulates translation. It can bind to, and thus recruit, several proteins that affect translation,^[19] one of them is initiation factor-4G with in turns recruits the 40S ribosomal subunit.^[21] A poly(A) tail is not a requirement for translation however.^[22]

Deadenylation

After export to the cytoplasm, the poly(A) tail of most mRNAs gradually gets shorter in eukaryotic somatic cells, and mRNAs with shorter poly(A) tail are translated less and degraded sooner.^[23] It can take many hours before an mRNA is degraded.^[24] Deadenylation can be accelerated by microRNAs complementary to the 3' untranslated region of an mRNA, this deadenylation initiates degradation of the mRNA.^[25] In the immature egg cells, mRNAs with shortened poly(A) tails are not degraded, but are stored without being translated. They are then activated by cytoplasmic polyadenylation after fertilisation, during egg activation.^[26]

Alternative polyadenylation

Results of using different polyadenylation sites on the same gene

The selection of which poly(A) site is used depends on the expression of the proteins that take part in polyadenylation. For example, the expression of CstF-64, a subunit of cleavage stimulatory factor (CstF), increases in macrophages in response to lipopolysaccharides (a group of bacterial compounds that trigger an immune response). This results in the selection of weak poly(A) sites and thus shorter transcripts, removing regulatory elements in the 3' untranslated regions of mRNAs for defense-related products like lysozyme and TNF-?. These mRNAs then have longer half-lives.^[28]

RNA-binding proteins other than those in the polyadenylation machinery can also affect whether a polyadenyation site is used or not in a cell,^[29] as can DNA methylation near the polyadenylation signal.^[30]

Tagging for degradation

For many non-coding RNAs, including tRNA, rRNA and snRNA, polyadenylation is a way of marking the RNA for degradation by the exosome in eukaryotes.^[31][32] This targets misfolded RNAs in particular.^[33] Many bacterial RNAs, both mRNAs and non-coding RNAs, are also polyadenylated to promote degradation, by the degradosome.^[34]

In eukaryotes, the polyadenylation of aberrant non-coding RNAs is done by the TRAMP complex, which adds an around 40 nucleotides long tail to the 3' end.^[35] Similarly, poly(A) tails in the bacterium E. coli, synthesised mainly by polyadenylate polymerase I, are 10?40 nucleotides long.^[36]

Evolution

Polynucleotide phosphorylase is an enzyme that is part of the bacterial degradosome and archaeal exosome (two closely related complexes that recycle RNA into nucleotides). It existed already in the last universal common ancestor. This enzyme degrades RNA by attacking the bond between the 3'-most nucleotides with a phosphate, breaking off a diphosphate nucleotide. This reaction is reversible, and so the enzyme can also extend RNA with more nucleotides. The tails added by polynucleotide phosphorylase are very rich in adenine. The choice of adenine is most likely the result of higher ADP concentrations than other nucleotides as a result of using ATP as an energy currency, making it more likely to be incorporated in this tail in early lifeforms. It has been suggested that the involvement of adenine-rich tails in RNA degradation prompted the later evolution of polyadenylate polymerases (the enzymes that produce poly(A) tails with no other nucleotides in them).^[37] Polyadenylate polymerases in both bacteria and eukaryotes have separately evolved from CCA-adding enzyme, which is the enzyme that completes the 3' ends of tRNAs. Its catalytic domain is homologous to that of other polymerases.^[31] Some lineages, like archaea and cyanobacteria, never evolved polyadenylation.^[37]

References

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