Post-transcriptional modification

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RNA Processing ( is also post-transcriptional modification) is modification of RNA after transcription. Biological significance:
a) RNA acquires additional structural features, b) diversification of signal from gene, c) point of modulation of expression, d.) quality control

tRNA and rRNA processing: in bacteria and yeast some tRNA and rRNA are transcribed together and then cleaved.   

rRNA is digested by endonucleases and exonucleases. Exonuclease is non sequence specific and endonuclease is sequence specific.  

tRNA processing: self splicing (removal of introns). CCA sequence is added to several bacterial tRNAs and is the sequence where the amino acid is added. 

Nucleotide modification occurs in functional domains of tRNA and rRNA. Mutation of the enzyme dyskerin involved in nucleotide modification leads to skin and bone malfunction.

mRNA 

- capping is essential for efficient elongation and termination and protects 5' from degradation. There are 3 steps in RNA capping: first removal of 5' phosphate then addition of GMP and then methylation of added guanine. 

- Poly-adenylation provides stability to mRNA (protection from exonuclease). Approximately 200 Adenosine are added at the 5' end. Alternative polyadenylation sites results in different proteins (e.g. immunoglobulins with 'earlier' polyadenylation site do not code for membrane anchoring domain). 2 steps; cleavage of RNA at specific site and addition of Adenosines.

Capping and polyadenylation depend on the phosphorylation of C-terminal domain (CTD) of RNA pol II.

RNA splicing = transesterification. Group I introns: Predominantly self splicing. C3-OH esterification. First step by free G nucleotide. Defined by 3D RNA structure. G-U wobble at splice sites (5' Exon 1 and 3' Exon 2). Group 2 introns: Internal nucleotide initiates reaction. C2-OH attack. Intron in 'lariat' branched form. Some genes have many introns.In the spliceosome, proteins are needed to regulate this process. 5' and 3' ends of splice sites need to be recognised. Exons need to be joined in the correct order. The spliceosome is the machinery that regulates this process (joins exons and removes introns). It is not fully understood.   

Alternative splicing. One gene generates multiple transcripts and thus multiple proteins (75% of genes). The transcript can include different exons, different translation start site or poly-adenylation sites and it can also include non-translated regions that contribute to stability or translocation of mRNA. 

Elucidation of splicing mechanism: Exon definition?? Intron definition?? 

Co-transcriptional splicing: Many splicing events occur while the RNA is being transcribed and the process is aided by marker proteins.

RNA editing: modification of nucleotides in the RNA sequence. Nucleotides are converted (A to ''G'', C to U). Adenosine -> Inosine.  Inosine recognised as guanidine. Cytidine -> Uridine. Nucleotides can also be insterted or deleted (U). The resulting protein may have different amino acid (point mutation). Insertion of stop codon. RNa editing can be tissue specific.RNA editing - excellent detailed site

RNA degradation is an important process for regulating gene expression (removing mRNA removes template for protein synthesis). RNA structure contribute to stability (5' cap, polyadenylation, loops). Some RNAs contain A-U rich regions that cause instability (e.g. c-fos  (viral mutant v-fos does not degrade and cause cancer)). Endo/exo-nucleases are involved in RNA degradation 

prokaryotes: pyrophosphate hydrolase -> endonuclease -> 3' to 5' exonuclease and poly-A destabilising in prokaryotes -> degradation)

eukaryotes: endonuclease -> poly-A stabilizing in eukaryotes -> either: 3' to 5' exonuclease or decapping enzyme -> 5' to 3' exonuclease

Short RNAs are involved in stability of full length RNA. Fragments of RNA are used to hybritize to complementary RNA sequence which are cleaved. Short RNAs include: siRNA (small interfering RNA) that is part of defensive mechanism against foreign RNA from viruses (sequences extracted from foreign RNA), miRNA (micro RNA) that is coded by the genome and regulate gene expression by fascilitating degradation of mRNAs. These are now exploited in research and as therapeutic strategy.

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