Transcription of messenger RNA (mRNA) from protein encoding genes and its translation by ribosomes into proteins is key to human biological function.
mRNA is written in the 5'→ 3' direction (as it is synthesised by RNA polymerase). It is single stranded and can form complex loops and other shapes. It is characterised by (see diagram) having:
- 5' methylguanosine cap. This is synthesised in four steps:
- Phosphate is cleaved from the end of the pre-mRNA transcript by a phosphatase
- Guanylyl transferase (RNGTT,HCE1,CAP1A) adds guanosine (fromGTP)to the end of transcript (using an unusual 5' to 5' linkage).
- Guanosine is then 7-methylated, with possibly some riboses at 5’ end methylated as well.
- The cap binds to cap binding complex (CBC, NCBP2, NIP1) which is necessary for export from the nucleus. Presumably it also protects mRNA from degradation which is the fate of single strand mRNA like transcribed sequences which do not have the cap.
- 5' UTR region. (5' untranslated region) This contains about 170 nucleotides including the RBS (ribosome binding site) and finishes with the start codon copied on DNA transcription (AUG – methionine)
- The coding region which has been assembled by RNA extron splicing, with typically 70% of the initial transcript intron RNA having been excised in man
- 3'URT starting from one of the three possible stop codon such as UAG
- Poly-A tail which is added to almost all mRNAs post-transcriptionally. The pre-mRNA transcript has an AAUAAA sequence (and downstream, removed at cleavage GU-rich region) that is recognised by CPSF (cleavage and polyadenylation specificity factor), a 4 subunit protein. This recruits cleavage stimulating factor F (CStF), an endonuclease, on binding to AAUAA. CStF (of which there are 3 in man) cleaves the transcript at a CA sequence. Poly-A polymerase, which is regulated by phosphoralation then adds up to 200 adenosine residues, which are bound by poly-A binding protein (PABP). In man there are 3 PABPs - PABP1, (PABPC1), inducible PABP (iPABP, PABPC4), and PABP3 (PABPC3) - which are important both because they remain attached to the RNA, perhaps being permissive to nucear export and because they specify how much poly-A is added which determines the half life of the mRNA in the cytosol. There appear to be two poly-A polymerases in man with the second being possibly specific and permissive to spermatogensis
During the pre-processing in the nucleus that results in these characteristics numerous protein factors can also be added which modify the translation process on the ribosome. But even before this is the process of transcription. In man this is done by RNA polymerase II which is not only an immensely complex protein in its own right, it is associated with numerous promotors and requires a helicase to work. Much of the pre-processing of pre-mRNA occurs concurrently to transcription by RNA polymerase II. As it is transcribed, it is complexed by small nuclear ribonucleoproteins (snRNPs, also known as 'U' particles) important in splicing. In man these are the U1, U2, U4/U6 and U5 ribonuclear protein complexes, made up of several of the 10 or more ribonucleoproteins and small nuclear RNAs (snRNAs) of the U family . Some splice out introns, others label the introns and exons. There may be 0 to 50 introns, although a typical pre-mRNA gene has 4 introns within 5 exons with the introns making up to 80% of the pre-RNA. Within the introns can be cytoplasmic regulators such as intron miRNA). The process of splicing is most obviously justified with immunogobulins where the multiple copies possible allow separation between the membrane receptor form of the immunoglobulin that stimulates soluble immunoglobulin formation from shorter transcripts.
Transport out of the nucleus of mRNA is through evolutionary conserved nuclear pore complex export proteins NXF1 (TAP) and NXT1 in man. The mRNA can have targeting sequences in the 3' UTR (untranslated region)which direct the mRNA to specific places in the cytoplasm. Examples include:
- Vasopressin mRNA direction to dendrites by a 400 nucleotide long sequence binding a PABP which in turn binds to cytoskeletal motors.
- Calmodulin dependent protein kinase II mRNA Y element binds to TB-RBP (testis-brain RNA binding protein)
This takes place with RNA interference by miRNA and protein regulation such as the endonucleases that chop up the poly-A tails of the proteins regulating iron metabolism. Further the mRNAs for transferrin blocks endonuclease binding while ferritin mRNA can block the RBS.
Translation takes place via ribosomes. It is a very complex process, involving t-RNA and multiple initiator and promotor proteins. From the point of view of the mRNA an initiation factor, eIF4A helicase, is important as it unwinds any hairpin loops in the mRNA, so exposing the start codon (AUG), and the proteins eIF4E and eIF4G by binding the mRNA CAP to the PABP on the poly-A tail are believed to promote ribosome efficiency as the ribosome can start again once its finished, without really dissociating from the mRNA.
Diseases of mRNA processing
The commonest acquired disease is SLE with anti-snRNP antibodies obviously having the potential to ruin intron excision.
While much of basic mRNA processing is presumably too critical to allow non-lethal mutation many of the transcription regulatory proteins, and presumaly miRNA etc are open to genetic disease or malignant transformation/deregulation. Some associations are:
- C-terminal domain of subunit A, phosphatase of subunit 1 (CTDP1, FCP1) mutation a protein phosphatase whose only known substrate is the phosphorylated serine residues of the carboxy-terminal domain of the largest subunit of RNA polymerase II causes congenital cataracts with facial dysmorphism and neuropathy, an autosomal recessive disorder.
- Transcription factor 3(TCF3, E2A) abnormalities in acute lymphoblastic leukaemia
- TWIST – Saethre-Chotzen syndrome and malignancy
- Transcription factor (TCF1) – MODY-3
- Prophet of PIT1, paired-like homeodomain transcription factor (PROP1) – combined pituitary hormone deficiency (CPHD)
- mRNA-binding proteins:
- Fragile X Mental Retardation protein (FMRP) is involved in translation and mutations cause fragile X mental retardation syndrome or the fragile X tremor/ataxia syndrome.
- Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1, CRDBP) in malignancy such as breast cancer
- Insulin-like growth factor 2 mRNA-binding protein 2 ( IGF2BP2, IMP2) in malignancy such as adenocarcinoma
- Insulin-like growth factor 2 mRNA-binding protein 3 ( IGF2BP3, IMP3, KOC1) in malignancy such as pancreatic cancer
- pre-mRNA splicing
- Precursor mRNA-processing factor 31 (PRPF31) mutations cause an autosomial dominant form of retinitis pigmentosa
- Precursor mRNA-processing factor 8 (PRPF8) mutations cause an autosomial dominant form of retinitis pigmentosa-13
- Precursor mRNA-processing factor 3 (PRPF3) mutations cause a form of retinitis pigmentosa-18
- mRNA editing enzymes
- Apolipoproten B mRNA-editing enzyme, Catalytic polypeptide-like (APOBEC) – excess expression in malignancy
- ↑ Kashiwabara S, Noguchi J, Zhuang T, Ohmura K, Honda A, Sugiura S, et al. Regulation of spermatogenesis by testis-specific, cytoplasmic poly(A) polymerase TPAP. Science 2002;298:1999-2002. (Direct link – subscription may be required.)
- ↑ Kalaydjieva L. Congenital cataracts - facial dysmorphism - neuropathy. Orphanet journal of rare diseases 2006;1:32. (Direct link – subscription may be required.)
- ↑ Nguyen H, Rendl M, Fuchs E. Tcf3 governs stem cell features and represses cell fate determination in skin. Cell 2006;127:171-83. (Direct link – subscription may be required.)
- ↑ Seifert G, Kress W, Meisel C, Henze G, Seeger K. Genetic investigations of Saethre-Chotzen syndrome presenting with renal cell carcinoma. Cancer genetics and cytogenetics 2006;171:76-8. (Direct link – subscription may be required.)
- ↑ Turton JP, Mehta A, Raza J, Woods KS, Tiulpakov A, Cassar J, et al. Mutations within the transcription factor PROP1 are rare in a cohort of patients with sporadic combined pituitary hormone deficiency (CPHD). Clinical endocrinology 2005;63:10-8. (Direct link – subscription may be required.)
- ↑ Davidovic L, Huot ME, Khandjian EW. Lost once, the Fragile X Mental Retardation protein is now back onto brain polyribosomes. RNA biology 2005;2:1-3.
- ↑ Nielsen J, Christiansen J, Lykke-Andersen J, Johnsen AH, Wewer UM, Nielsen FC. A family of insulin-like growth factor II mRNA-binding proteins represses translation in late development. Molecular and cellular biology 1999;19:1262-70.
- ↑ Beale RC, Petersen-Mahrt SK, Watt IN, Harris RS, Rada C, Neuberger MS. Comparison of the differential context-dependence of DNA deamination by APOBEC enzymes: correlation with mutation spectra in vivo. Journal of molecular biology 2004;337:585-96. (Direct link – subscription may be required.)