Physiological and Experimental Roles for Non-coding RNAs

Physiological and Experimental Roles for Non-coding RNAs

1. Protein coding potential of genomes

   • ~5% of cellular RNA content is messenger RNA (mRNA), ~80% is ribosomal RNA, other 15% is non-coding RNA
     • ncRNAs
       • miRNAs & siRNAs
         • RNA interference (RNAi), gene silencing mechanism induced by double stranded RNA - recognises complementary mRNA and recruits protein complexes for gene silencing
           • Introducing double stranded RNA (dsRNA) into Drosophila & C.elegans, induces silencing of complementary mRNA. Processed to make short interfering RNAs (siRNA), induces gene silencing and recruits RNA induced silencing complex (RISC)
           • miRNA - gene silencing mechanisms
             • RISC seeks out its target mRNAs by searching for complementary nucleotide sequences. Facilitated by the Argonaute protein, component of RISC. Pairing often occurs at the 3' UTR region of target mRNA
             • Method 1: If base pairing is extensive then the mRNA is cleaved by the Argonaute protein, removing the poly-A tail exposing it to exonucleases. Once cleaved the RISC with its miRNA is released and can seek out additional mRNAs. So can act on more than one mRNA. Can be thought of as 'guide' sequences that bring destructive nucleases into contact with specific mRNAs.
             • Method 2: If base pairing is less extensive between miRNA and mRNA, Argonaute does not slice the mRNA; translation of the mRNA is repressed and is shuttled to P-Bodies where it eventually undergoes poly-A tail shortening, decapping and degradation.
         • Biosynthesis of miRNA
           • miRNA genes occurwithin introns of protein coding genes. 1. long dsRNA transcribed by Pol II. 2. Folds into hairpin and cut by drosha. 3. Moved into cytoplasm by exportin 5. 4. Processed to active form of mRNA by DICER
         • Role of miRNAs
           • Advantages; Rapidly turn off expression of specific genes, co-ordinate regulation of complex processes. miRNAs in neural development e.g. mir-124
             • Repressor Element-1 Silencing Transcripton Factor (REST) regulates the expression of mRNAs and miRNAs . It regulates miR-124 which blocks TF Sox9
         • Short interfering RNA (siRNA) - chemically synthesised to artificially induce gene silencing. shRNA = short hairpin RNA expressed from a plasmid.
           • siRNA to deplete REST - can quantify bu using DNA microarray.
             • siRNA Libraries
               • siRNA or shRNA libraries are used in genetic screens to target whole genome or one protein family or a specific pathway
       • Small stable RNAs
         • Transfer RNA (tRNA) - translation, Small nuclear RNA (snRNA) - splicing, Small nucleolar RNA (snoRNA) - rRNA processing
       • Long ncRNAs
         • Have properties of mRNA but DO NO code for proteins. e.g. Natural antisense transcripts (NATs) expressed from opposite strand for 15-25% of genes. Specific secondary structure, interact with chromatin modifying enzymes, coordinate epigenetic modification of nearby chromatin.

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