MicroRNA Silencing Through RISC Recruitment of eIF6

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MicroRNA Silencing Through RISC Recruitment of
eIF6
By Thimmaiah P. Chendrimada, et al.

• William Hung and Jing Jin

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Inhibition of Gene Expression through RNA
interference
RNA interference (RNAi) is a mechanism that
inhibits gene expression. There are two pathways

• Small interfering (si)RNAs short dsRNAs of 20-25
  nucleotides in length. They typically base-pair
  perfectly to the target mRNA and induce mRNA
  cleavage.
• Micro (mi)RNAs single-stranded, non-coding RNAs
  of 22 nucleotides in length. They are typically
  only partially complementary to the mRNA target
  and inhibit protein translation. When the miRNA
  is perfectly complementary to the mRNA target, it
  can also trigger cleavage.

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miRNA and siRNA Triggers RISC to Cleave mRNA

• RISC (RNA-induced silencing complex) is a multi
  protein complex that contains Dicer, TRBP, and
  AGO2 (argonaute 2)

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Question

• What is the repressive mechanism exerted by
  miRNAs that partially base-pair to target mRNA
  sequences?
• What are the specific factors required for such
  miRNA function?

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A Larger TRBP-Containing Complex

• previously identified Dicer-TRBP-AGO2 complex
  (RISC) was 500 kDa.
• Using flag-tagged TRBP, a 2 MDa TRBP-containing
  complex was found. (fraction 16)

Gel filtration SDS-PAGE w/ silver staining of
the 2 MDa fraction.
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eIF6 is a Part of the Large TRBP-Containing
Complex
The 2 MDa TRBP-containing complex has two
components 500 kDa RISC and a TRBP-associated
complex that contains

• eIF6
• Proteins from 60S ribosome subunit
• (but not from 40S)
• RNA helicase MOV10

Western blot analysis.
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eIF6 is a 60S Ribosome-Associated Factor

• The eukaryotic ribosome (80S) consists of a small
  subunit (40S) and a large subunit (60S).
• eIF6 is a 60S ribosome-associated factor.
• eIF6 prevents the assembly of the ribosome by
  binding to the large subunit 60S.

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5 end of miRNA Needs to be Complementary to mRNA
to Repress Translation

• Reporter constructs Renilla luciferase mRNAs
  with one or two binding sites for let-7b (a
  naturally-produced, classic miRNA).
• The mutant construct has a C-insertion in the 5
  end of miRNA

The mutant has higher luciferase activity because
the miRNA does not bind well.
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eIF6 is Needed for miRNA-induced Repression

• Increased activity for WT when miRNAs are
  silenced using asRNAs.
• Increased activity for WT when eIF6 is depleted
  using siRNAs.
• No change for the mutant construct.

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eIF6 Inhibits the Assembly of RISC and 60S
Ribosome Subunit

• eIF6 depletion leads to a loss of free 60S
  ribosome subunits and an appearance of a
  shoulder on the 80S subunit.
• ?60S is associated with higher molecular mass
  complexes (RISC) in the absence of eIF6.

mRNA of the housekeeping gene GAPDH is not
affected because the miRNA is not specific to
this mRNA.
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miRNA-induced Gene Repression Decreases mRNA
Levels

• Renilla luciferase mRNAs with two let-7b sites in
  the 3 UTR.

• mRNA is detected
• When the miRNA binding site is not inserted. (1)
• When the binding site is mutated. (lane 2)
• When eIF6 is depleted. (lane 4)
• mRNA is not detected when the binding site is
  introduced. (lane 3)
• ? mRNAs are not cleaved but are not accumulated.

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Question 2 - Is the function of eIF6 conserved
in other organisms?

• Experimenters tested role of eIF6 on lin-4 miRNA
  in C. elegans (nematode)
• Lin-4 miRNA negatively regulates expression of
  LIN-14 and LIN-28 proteins by base pairing to
  their mRNA precursors

• Experimenters compared wildtype cells to two
  types
• of mutant cells
• 1. lin-4(e912lf) expresses no lin-4 miRNA
• 2. lin-14(n355gf) lacks base pairing sites for
  lin-4 miRNA

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eIF6 regulates lin-4 miRNA targets in C. elegans
Western Blots (Proteins)

• Tubulin/Actin used as loading controls
• eif-6 RNAi resulted in decreased expression of
  eIF-6
• Wildtype cells subjected to eif-6 RNAi resulted
  in increased expression of LIN-14 and LIN-28
  proteins (Fig. 3b/3c)
• lin-4(e912if) mutant cells subjected to eif-6
  RNAi had no effect in LIN-14 and LIN-28
  expression (Fig. 3b/3c)
• Similarly, lin-14(n355gf) mutant cells subjected
  to eif-6 RNAi had no effect in LIN-14 and LIN-28
  expression (Fig. 3c)
• Therefore, lin-4 is the primary negative
  regulator of LIN-14 and LIN-28 expression
• eIF6 must regulate LIN-14 and LIN-28 expression
  via miRNA regulation

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eif-6 regulates lin-4-responsive mRNA in C.
elegans
Northern Blots (mRNA)

• Actin was used as a loading control
• No difference in lin-4 miRNA levels in the
  addition and absence of eif-6 RNAi in wildtype
  cells (Fig. 4a)
• However, eif-6 RNAi caused increased levels of
  lin-14 and lin-28 mRNA in wildtype cells (Fig.
  4a)
• eif-6 RNAi did not cause a significant increase
  in levels of lin-14 and lin-28 mRNA in
  lin-4(e912lf) and lin-14(n355gf) (Fig. 4b)
• unc-22 RNAi used as a control (Fig. 4c)
• These results indicate that over-expression of
  lin mRNA is miRNA dependent, but ultimately
  controlled by eIF6

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eIF-6 is a key lin-4 miRNA-dependent regulator of
lin-14 and lin-28 in C. elegans
Northern Blots (mRNA)

• Experimenters tried to identify time point where
  lin-4 miRNA directs translational repression
• In absence of eif-6 RNAi, lin-4 miRNA first
  present at 12 h and reaches maximum at 16 h
• After 20h, no difference in lin-4 miRNA in
  presence and absence of eif-6 RNAi
• In presence of eif-6 miRNA, slight increase in
  pre-rRNA precursors, consistent with earlier
  yeast studies

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Discussion - Conclusions

• eIF6 is associated with the RISC complex and the
  60S ribosome, and it inhibits the assembly of the
  two complexes.
• eIF6 mediates miRNA-induced gene silencing in
  human cells
• eIF6 regulation of miRNA-targets is conserved in
  C. elegans

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Discussion Future Directions

• Determine other factors that that regulate the
  lin-14 gene in C. elegans model system
• Determine if eIF6-mediated gene silencing is
  conserved in other model systems
• Elucidate the exact role of processing bodies (P
  bodies) in gene silencing do components of P
  bodies execute silencing or degrade mRNA?