miRNA

Question 1

function - miRNA

Answer 1

• miRNA are short noncoding RNAs that once made, base-pair with specific mRNAs and regulate their stability and their translation
  • Humans, for example, express more than 400 different miRNAs
  • These appear to regulate at least one-third of all human protein-coding genes.
• A single miRNA can act catalytically to destroy/inhibit many complementary mRNAs

Question 2

production -miRNA

Answer 2

1. The miRNA precursors are synthesized by RNA polymerase II and are capped and polyadenylated.
2. They then undergo a special type of processing, cropping (by drosha in nucleus) and dicing (by dicer)
3. The miRNA is then assembled with a set of proteins to form an RNA-induced silencing complex or RISC.
4. The RISC seeks out its target mRNAs by searching for complementary nucleotide sequences
   • This search is greatly facilitated by the Argonaute protein, a component of RISC, which displays the 5’ region of the miRNA so that it is optimally positioned for base-pairing to another RNA molecule
   • In animals, the extent of base-pairing is typically seven nucleotide pairs
5. Once an mRNA has been bound by an miRNA, several outcomes are possible.
   a. If the base-pairing is extensive, the mRNA is cleaved by the Argonaute protein, effectively removing its poly-A tail and exposing it to exonucleases
    RISC/miRNA complex is released, and it can seek out additional mRNAs.
   b. If the base-pairing between the miRNA and the mRNA is less extensive, translation of the mRNA is repressed and the mRNA is destabilized.
    This effect is associated with shortening of the poly-A tail and the movement of the mRNA to cytosolic structures called processing bodies
    Here the mRNAs are sequestered from ribosomes and eventually decapped and degraded.

Question 3

function - siRNA

Answer 3

• short interfering RNA
• dsRNA triggers iRNA system which breaks down the dsRNA into siRNA
• used in RISC which leads to mRNA cleavage
• used in RITS which leads to transcriptional repression (methylation of DNA and histones)

Question 4

production - siRNA

Answer 4

• The presence of double-stranded RNA in the cell triggers RNAi by attracting a protein complex containing Dicer, the same nuclease that processes miRNAs
  1. This protein complex cleaves the double-stranded RNA into small fragments called small interfering RNAs (siRNA).
  2. These double-stranded siRNAs are then bound by Argonaute and other components of the RISC, as we saw above for miRNAs, and one strand of the duplex RNA is cleaved by Argonaute and discarded.
  3. The single-stranded siRNA molecule that remains directs RISC back to complementary RNA which leads to cleavage of the mRNA since the sequence is 100% complementary
• RNA interference machinery can selectively shut off synthesis of the target RNAs.
  1. siRNAs produced by the Dicer protein are assembled with a group of proteins (including Argonaute) to form the RITS (RNA-induced transcriptional silencing) complex.
  2. Using siRNA as a guide sequence, this complex binds complementary mRNA as they emerge from a transcribing RNA polymerase II
  3. The RITS complex attracts proteins that covalently modify nearby histones and eventually directs the formation of heterochromatin to prevent further transcription initiation.
  4. In some cases, the RITS complex also induces the methylation of DNA and histones to further stop transcription

Question 5

function - shRNA

Answer 5

• A short hairpin RNA is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference
• Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors.
• shRNA is an advantageous mediator of RNAi in that it has a relatively low rate of degradation

Question 6

production - shRNA

Answer 6

1. Once the vector has integrated into the host genome, the shRNA is then transcribed in the nucleus by polymerase II or polymerase III depending on the promoter choice.
2. The product mimics pri-microRNA (pri-miRNA) and is processed by Drosha.
3. The resulting pre-shRNA is exported from the nucleus by Exportin 5.
4. This product is then processed by Dicer and loaded into the RNA-induced silencing complex (RISC). The sense (passenger) strand is degraded.
5. The antisense (guide) strand directs RISC to mRNA that has a complementary sequence. In the case of perfect complementarity, RISC cleaves the mRNA.

Question 7

Protein kinase R reaction

Answer 7

• Protein kinase R (PKR) is an interferon-induced kinase that plays a key role in the innate immunity response to viral infection.
• The enzyme is synthesized in a latent state but it is activated by binding dsRNA to undergo autophosphorylation at multiple serine, threonine and tyrosine residues.
• The most well characterized cellular substrate of PKR is the alpha subunit of eukaryotic initiation factor eIF2.
  • Phosphorylation of eIF2 inhibits protein synthesis in virally-infected cells.
  • Thus, production of dsRNA during viral infection results in PKR activation and subsequent inhibition of viral and host protein synthesis.
• PKR activates FADD –> apoptosis
• PKR inhibits AKT/mTOR –> stops growth
• PKR causes NFkB activation –> inflammation
• The importance of PKR in antiviral defence is underscored by the large number of viruses that encode PKR inhibitors.
• PKR also functions in the control of cell growth and proliferation and as a tumour suppressor protein