RNA interference and microRNAs

notes

• RNA interference (RNAI) has made a major emergence in the biomedical sciences only in the last decade or so 

• Research into RNA interference pathways eventually led to the characterisation of microRNAs now known to be fundamental players in cellular mRNA translation regulation 

the small interfering RNA (siRNA) pathway

• Thought to have evolved as a viral defence mechanism as early as in plants 

• The pathway consists of machinery that can generate small double-stranded RNAs or siRNAs from viral genome and then use them to destroy viral mRNAs 

• Double stranded RNA genome (retrovirus) 

• DNA copy of RNA Is made 

  • Gets incorporated into the plant genome 

• Genome is expressed (mRNA made) 

  • Makes viral parts 

• Foreign genetic material is recognised 

  • Some of the copies of double stranded RNA are degraded into small RNAs (sIRNA) by Dicer 

  • They interact with RISC complex (RNA induced silencing complex) 

  • One strand is rejected 

  • One strand (guide strand) is retained within the complex) 

  • Protein RNA complex scans cell for any complimentary sequences (will only be viral sequences) 

• If there is a perfect matching of sequence 

  • Enzymatic components of the RISC complex degrade target 

• Viral DNA degraded 

  • Viral particles no longer produced 

Dicer (enzyme)

• Dicer generates the small double stranded siRNAs from the double-stranded viral RNA genome 

• The small siRNAs need to be in a very precise molecular configuration in order to be accepted by the RISC complex 

  • 22 nucleotides of RNA molecule 

  • With 2 nucleotide 3' overhangs on each end 

• Dicer enzyme is composed of multiple domains 

  • PAZ domain 

    • Locks onto 3' end of RNA 

    • Anchors the RNA 

  • Ruler helix 

    • Measures out 22 nucleotide length  

  • RNA domain 

    • Perform incision 

    • Not perfectly aligned 

    • One enzyme performs incision, other performs the incision 2 extra nucleotide away 

    • Forms the other overhang 

RISC

 

• The RNA induced silencing complex mediates potent enzymatic destruction of the viral mRNA copies 

• Will only take up RNA generated by Dicer 

Ago

• Argonuate (Ago/Ago2) is the major component of the RISC complex and carries out its critical function 

• Has multiple domains 

  • MID 

    • Binds to 5' end 

  • PIWI 

    • Contains enzymes 

    • Responsible for mRNA cleavage 

  • PAZ 

    • Binds to 3' end of RNA 

  • N-terminal 

    • Unwinds RNA 

• Only once a perfect match has been found then will enzymatic components be reached 

  • Released from where 

    • Piwi domain 

microRNAs

• Higher organisms such as vertebrates have evolved alternative more effective and comprehensive means of viral defence 

• However we surprisingly find that the siRNA pathway machinery is still produced in vertebrate/mammalian cells 

• The pathway was hijacked by endogenously produced miRNAs expressed from vertebrate genomes 

• Several thousand miRNA genes have been identified in our genome 

  • At least one for each protein coding gene 

• These genes encode lager miRNA-precursor RNAs which are then processed by cellular enzymes into the much shorter mature miRNAs

• The mature roughly 22 nucleotide long miRNAs are used by the cell to exert control over mRNA translation 

pre-miRNA

• Pre-miRNA recognised by Dicer complex 

• How is hairpin portion cleaved off? 

  • PAZ domain binds to 3' end 

  • Cleaved near the base of the stem 

  • Terminal/hairpin loop is cut off 

  • 22 nucleotides long as it is cleaved by dicer 

• Due to configuration, it will interact with RISC complex 

  • One strand is retained in complex 

  • Can scan cytoplasm for sequency complementarity 

• Matching of sequence of miRNA to mRNA is never perfect 

  • Thus do not get degradation of mRNA 

• Seed sequence = perfect match in 5' end of miRNA 

• Pathway is to temporarily halt translation 

  • Can inhibit elongation of ribosome 

• If cell wants to shut of translation permanently it can express mRNA exactly complementary to the miRNA 

piRNAs

• Piwi-interacting RNAs are expressed almost exclusively in germ line cells 

• In mammalian germ line cells, bursts of retrotransposon activity can be observed and piRNAs are employed to control/supress this activity 

  • To promote genetic diversity  

  • Can cause sections of genetic material to swap around 

  • Eventually needs to be controlled (could become mutagenic) 

action of piRNAs

• Line 1 gene expressed into line-1 RNA 

• piRNA cluster gene is located on opposite strand to line-1 gene 

  • Complementary to line 1 gene 

  • Also expressed into RNA 

• Piwi gets hold of piRNA precursor 

  • Starts cutting it into smaller molecules 

• Retains smaller pieces as mature piRNA 

  • As it is complementary to line-1 RNA it can form an interaction 

• Interaction occurs in nucleus 

• As line-1 emerges from polymerase 

  • Newly synthesised 

• Piwi is recruited to gene 

• Recruits other proteins 

  • DNA methyltransferases 

    • Prevents binding of transcription factors 

  • Histone methyltransferases 

    • Adds methyl to histones 

    • Reduces gene expression