RNA

• RNA Interference (RNAi)


  Recently emerged in molecular genetics, research into RNAi pathways led to the characterisation of miRNAs, which are fundamental players in mRNA translation regulation
• Major pathways


  • siRNA
  • miRNA
• Small interfering RNA (siRNA) pathway


  1. Thought to have evolved as a viral defence mechanism in plants

  2. Consists of machinery that can generate small double-stranded RNAs (siRNAs) from viral genomes and use them to destroy viral mRNAs
• Dicer enzyme


  • Degrades the double-stranded viral RNA genome into small pieces (siRNAs)
  • The siRNAs need to be in a precise molecular configuration to be accepted by the RISC
  • 2-nucleotide overhangs on both of their 3' prime ends
  • PAZ domain: locks on to the 3' prime end
  • Ruler helix domain: maintains a length of 22 nucleotides between the PAZ domain and RNase domains
  • RNase domains: make the incisions 22nt upstream of the end of the molecule, 2 nucleotides offset to ensure the 2-nt overhang is made
• RNA Induced Silencing Complex (RISC)


  1. RISC protein complex binds to the siRNA (only if they're in the correct configuration)

  2. One strand is ejected and degraded, the other is retained and is called the guide strand

  3. Guide strand + RISC complex scans the cytoplasm for sequence complementarity to the guide siRNA strand

  4. RISC senses 'perfect' base pairing, activating enzymatic activity to degrade the target viral mRNA

  5. Argonuate is the major enzymatic component, with RNA helicase activity, PAZ domain, MID domain, and PIWI domain with RNase activity
• Micro RNAs (miRNAs)


  • Higher organisms, such as vertebrates, have evolved alternative more effective means of viral defence, but the siRNA pathway machinery is still produced in mammals
  • The siRNA pathway was hijacked by endogenously produced miRNAs, expressed from vertebrate genomes
  • Involved in post-transcriptional regulation and RNA silencing
• miRNA biogenesis


  1. Micro RNA gene in our genome is transcribed into primary micro RNA (pri-miRNA)

  2. DROSHA complex recognises the hairpin containing the mature miRNA and cuts the base of the hairpin on the pri-miRNA strand, forming pre-miRNA + a 2-nt 3' prime overhang

  3. PAZ domain of a dicer enzyme locks on to the 3' overhang, its RNase domains will produce a 22nt strand, forming a miRNA (retained in RISC) /miRNA* duplex (ejected from RISC)
• miRNA-mRNA base pairing


  • Base pairing between miRNA and mRNA is never fully complementary, only complementary on the 5' end (seed sequence)
  • This leads to inhibition of ribosome elongation so translation cannot be carried out, providing a temporary shut-off to translation
• Piwi-Interacting RNAs (piRNAs)


  • Expressed almost exclusively in germ line cells
  • In mammalian germ line cells, bursts of potentially harmful retrotransposon activity are controlled / supressed by piRNAs
  • piRNA genes are on the complementary strand to LINE-1 genes, PIWI proteins recognise the piRNA precursors and fragment them into smaller pieces, PIWI protein retains the fragments (piRNA) and can mediate silencing of the transcribed transposon RNA
• piRNA-mediated silencing


  1. LINE-1 recognition occurs as it's being transcribed

  2. Interaction of LINE-1 and PIWI complex recruits DNA methyl-transferase enzymes, which methylate DNA, inhibiting transcription of the gene

  3. Histone methylation proteins can also be recruited, also inhibiting transcription of the gene
• Types of non-coding DNA sequences


  • Transcriptional regulation: promoters, enhancers, silencers, insulators
  • Non-coding RNA genes: make a functional RNA molecule
• Transcriptional regulation


  • Control where and to what level genes are expressed
  • All cells have the same genome but have different structure/function due to expression regulation
• RNA polymerases


  • RNA polymerase I: 5.8S, 18S and 28S rRNA genes
  • RNA polymerase II: all protein-coding genes + snoRNA, miRNA, siRNA, lncRNA and snRNA
  • RNA polymerase III: tRNA, 55S rRNA, some snRNA
• RNA polymerase II


  • A large 12 subunit protein that is recruited to target genes along with general transcription factors (GTFs) and regulatory proteins in a holoenzyme complex
• Cis-regulatory elements


  • Located on the same chromosome
  • Promoter: define the position where transcription begins (transcription start site)
  • Enhancers & silencers: enable a high level of accurate transcription, act like switches to turn a gene on or off
  • Transcription factors: read the sequence of cis regulatory DNA and can interact with GTFs and RNA polymerase II
  • Insulators: prevent regulation of adjacent genes, act in a position-dependent way, barrier elements prevent the spread of condensed chromatin into the actively transcribed region
• After the human genome project was completed, multiple international collaborations were established to identify and annotate all the functional DNA elements in the non-coding genome
• Encyclopaedia of DNA elements (ENCODE) project


  • Used many techniques, genome sequencing and computational analysis to map regulatory elements genome-wide
  • Found that 4.6% of bases are covered by a transcription factor binding-site motif
  • The genome expresses thousands of non-coding RNAs
  • Identified 70,000 regions with promoter-like features
• Long non-coding RNA (lncRNA)

  • 200+ nucleotides
  • Transcribed by RNA polymerase II, don't encode for protein, some can generate small peptides
  • Possess a polyA tail and 5' 7 methylguanosine cap
  • Fewer exons and shorter transcript length compared to mRNAs
  • More tissue-restricted expression than mRNAs
  • Located in the nucleus or cytoplasm
• Functional Annotation of the Mammalian Genome (FANTOM) project


  • Identified 28,000 high-confidence human lncRNA genes
  • Many lncRNA genes are co-expressed with nearby protein coding genes, suggesting a role in local transcriptional regulation
  • Most transcriptional enhancers generate a lncRNA
  • A subset of lncRNAs have emerged as a new class of gene expression regulators, acting in the nucleus or cytoplasm