The following points highlight the four main processes to produce mRNA from RNA. The processes are: 1. Splicing 2. Capping 3. Polyadenylation 4. RNA Editing.
Process # 1. Splicing:
This process takes place in the nucleus and involves the removal of noncoding intron sequences from pre-mRNAs to produce mature mRNAs in which the coding sequences, corresponding to the exons, are continuous. The mature spliced mRNA, an accurate template for protein synthesis, is then exported to the cytoplasm where it acts as a template for protein synthesis.
Splicing depends on the presence of signal sequences in the pre-mRNA. In almost all genes, the first two nucleotides at the 5′ end of an intron are GT and the last two at the 3′ end are AG. These are part of larger signal sequences present at the 5′ and 3′ ends of the introns. The complete 5′ signal sequence is 5′ AGGTAAGT 3′ and the 3′ sequence is 5′ YYYYYYNCAG 3′ (Y = pyrimidine, N = any nucleotide).
A branch point sequence is present in vertebrates, in the introns 10-40 bases upstream of the 3′ signal sequence. A more specific sequence 5′ UACUAAC 3′, occurs in introns of yeast. Splicing occurs in two steps (Fig. 16.8A). In the first step, the 2′ hydroxyl group of the adenine of the branch point sequence attacks the phosphodiester bond 5′ to the G of the GT (5′ splice site).
The bond is broken releasing the 5′ end of the intron and attaching it to the branch point sequence. The intron now forms a tailed loop structure called a lariat. In the second step, the 3′ end of the Intron is cleaved after G of the AG (3′ splice site), the intron is released and the two exon sequences are joined together.
Splicing is catalyzed by a group of molecules called small nuclear ribonucleoproteins (snRNPs) – U1, U2, U4, U5 and U6. These are composed of small RNA molecules rich in uracil, called U RNAs or small nuclear RNAs (snRNAs) that exist complexed with proteins. The U1 snRNP binds to the 5′ splice site and the U2 snRNP binds to the branch point sequence.
The remaining snRNPs, U5 and U4/U6, then form a complex with U1 and U2 causing the intron to loop out and the exons to be brought together. The combination of the prg-mRNA and the snRNPs is called the spliceosome and this is responsible for folding the pre-mRNA into the correct conformation for splicing (Fig. 16.8B).
The spliceosome also catalyzes the cutting and joining reactions that excise the intron and ligate the exons. Once splicing is completed the spliceosome dissociates.
Process # 2. Capping:
Eukaryotic pre-mRNAs are altered at their 5′ end by a modification known as capping which involves addition of the modified nucleotide, 7-methylguanosine. The cap is added by the enzyme guanyltransferase which joins GTP by an unusual 5′—- > 5′ triphosphate linkage to the first nucleotide of the mRNA.
Methyl transferase enzymes then add a -CH3 group to the 7-nitrogen of the guanine ring and, usually, to the 2′ hydroxyl group on the ribose sugar of the next two nucleotides. Capping protects the mRNA from being degraded from the 5′ end by exonucleases in the cytoplasm and is also a signal allowing the ribosome to recognize the start of a mRNA molecule (Fig. 16.9A).
Process # 3. Polyadenylation:
Most eukaryotic pre- mRNA are modified at their 3′ ends by the addition of a sequence of up to 250 adenines, known as a poly A tail. This modification is called polyadenylation and requires the presence of signal sequences in the pre-mRNA.
These consist of the polyadenylation signal sequence, 5′ AAUAAA 3′, which occurs near the 3′ end of the pre-mRNA. The sequence YA (Y = pyrimidine) occurs in the next 11-20 bases and a GU rich sequence is often present further downstream. A number of specific proteins recognize and bind these signal sequences forming a complex which cleaves the mRNA about 20 nucleotides downstream of the 5′ AAUAAA 3′ sequence.
The enzyme poly(A) polymerase then adds adenines to the 3′ end of the molecule. The purpose of the poly A tail may be to protect the mRNA from degradation of the coding sequence at the 3′ end by exonucleases (Fig. 16.9B).
Process # 4. RNA Editing:
Pre-mRNA may also undergo RNA editing in which the sequence of the pre- mRNA is altered by the insertion, deletion or substitution of bases. RNA editing was first identified in the mitochondrial gene in which the transcripts were found to be extensively modified by the insertion of uracil residues. RNA editing, may involve either the whole gene, i.e., pan-editing or just a few bases, i.e., minor editing.
Different types of RNA editing are:
(a) Base insertion/deletion type (Fig. 16.9C)
(i) U-insertion/deletion editing in the kinetoplastid, mitochondria is catalyzed by editosome through cleavage, addition or removal and ligation utilizing guide RNAs (gRNAs).
(ii) C-insertion/dinucleotide (GC, GU, CU, AA, AU) editing in mitochondria of slime moulds is achieved by slippery transcription.
(iii) G or A-insertion editing found in negative strand RNA viruses and Ebola virus respectively.
(b) Base substitution/modification type (Fig. 16.9D)
(i) C to U editing in plant mitochondria and chloroplasts, mammalian apo B, etc. involves transition due to deamination of cytosine by cytidine deaminase.
(ii) A to I (inosine) editing in glutamate receptor, hepatitis delta virus, etc. occurs through deamination of adenosine with the help of adenosine deaminases acting on RNA (ADARs) by specifically target- ting single nucleotides within partially double stranded pre-mRNAs.
(iii) A to G or U to A or U to G editing found in vertebrate mRNAs.
