BGM3055 Presentation on

Translational Repression and eIF4A2 Activity Are Critical for MicroRNA-

Mediated Gene Regulation

Presenters:Michael Thirlwell, KATE Wisdom Deebeke, and Mene Nkue.

Aims and Objectives:• To discuss the General concept of Translational

repression• To describe the methods used in this study• To analyse the results and evaluate the outcome of the

experiments

RNAi Timeline 1990: Introduction of gene copies into Petunia led to patchy plants.

1993: First miRNA discovered in C. elegans

1998: Injection of dsRNAs into C. elegans led to silencing of complementary genes

2000: Second miRNA discovered in C. elegans

2004: RNAi review paper claims: “Arguably the most important advance in biology in decades has been the discovery that RNA molecules can regulate the expression of genes.”

Novina et. al 1990

siRNA dsRNA can be produced by exogenous sources (e.g. virus) orfrom a transgene or a rogue genetic element.

dsRNA is cleaved into fragments (20-25 bp) by Dicer

RISC binds fragments and degrades sense strand

Remaining antisense strand hybridizes with mRNA

RISC degrades mRNA

miRNA Genome codes for transcription of pri-miRNAs which due to imperfect self complementarity fold into a hairpin

Drosha cleaves ends to form pre-miRNAs

Dicer cleaves into 21-22 bp single stranded miRNAs

miRNA forms miRNP which then binds mRNA imperfectly, and so inhibiting translation

Rarely hybridises perfectly and soleads to mRNA degradation

Translational Repression and eIF4A2 Activity Are Critical for MicroRNA-Mediated Gene Regulation

This paper studied the mechanism of miRNA(let-7) inhibition of mRNA

Found that unadenylated and polyadenylated mRNAs are repressed

Greater magnitude of repression when mRNA is polyadenylated

mRNA Destabilization follows Repression.

Discovered that “eIF4A2 plays a critical role early in the repression pathway”

Methods

Plasmid constructs:• Designed different plasmid constructs (pRL, pRL-Let-7

and pRL-Let-2) for different experiments:

• IRES for pRL-HCV/let-7, pRL-EMCV/let-7 and pRL-CrPV/let-7 • ß-globin 5’UTR or the (CAA)18

5’UTR• SiRNA generated against specific

translation initiation factor proteins• Used specific RE to digest plasmids

and insert the ‘’insert’’

Fig. Taken from Promega website (promega.co.uk)

Methods continued

In vitro transcription and polyadenylation

(with the exception of globin-pRL/Let-7 and CAA-pRL/Let-7)

– Linearised plasmid with BamHI followed by Agarose gel purification.

– Polyadenylation with poly(A) tailing kit

Fig. Taken from 5PRIME website (www.5prime.com)

Methods continued

• Different mRNAs transcribed:

Meijer et al., Science 340:82-5 (2013)

Schematic representation of RNA used for transfection.

RNA used in deadenylation study

IRES-containing RNA used for the transfection study

Schematic representation of RNA transcribed from globin and CAA18 plasmids

Methods continued Transfected plasmids intoHeLa, HEK293 or HCT cells

byLipofectamine2000, fugeneor Genejammer methods

Other assays include:• Luciferase assay (assayed for

Rluc relative to Fluc)• Immunoprecipitation (WB for

transfected eIF4A1/2)• RNA isolation, RT-qPCR and

PAT assays

Fig. Taken from: http://www.bocascientific.com

Translational Repression contributes to miRNA mediated gene regulation

Repression of translation by let-7 miRNA increases with time. CrPV: no initiation factor HCV: requires only eIF(2,3,5) eIF5b & Met tRNAi

EMCV: requires all initiation factor except 4E Translational repression is required for miRNA-mediated control elF4F is essential for repression mRNA destabilisation follows translational repression

Meijer et al., Science 340:82-5 (2013)

eIF4F is required for miRNA mediated mRNA repression

Beta globin 5’UTR unstructured(CAA)18 5’UTR (does not require unwinding) unstructured 5’UTR was refractory to miRNA-mediated repression, showed slight activation

showed that 4A is the implicated EIf4f required for miRNA-mediated translational repression

EIf4A2 is the only component of elF4F required for miRNA-mediated repression

siRNA was used to knockdown the expression of eIF4F components

after depletion of eIF4A2, only eIF4A2 and not eIF4A1 can restore repression

eIF4A2 is the key factor for miRNA-mediated gene silencing.

Conclusion Brief history of RNAi (miRNA and siRNA), biological

application

Repression of translation by Let-7 miRNA

Key methods used to achieve repression of mRNA translation by the miRNA Let-7

Requirement of translational repression and the eIF4A2 for miRNA-mediated gene control

References

• Meijer, H.A., Kong, Y.W., Lu, W.T., Wilczynska, A., Spriggs, R.V., Robinson, S.W., Godfrey, J.D., Willis, A.E. and Bushell, M. (2013) 'Translational Repression and eIF4A2 Activity Are Critical for MicroRNA-Mediated Gene Regulation', Science, 340(6128), pp. 82-85.

• Fabian, M.R., Sonenberg, N. and Filipowicz, W. (2010) 'Regulation of mRNA Translation and Stability by microRNAs', Annual Review of Biochemistry, 79(1), pp. 351-379.

• Fabian, M.R., Mathonnet, G., Sundermeier, T., Mathys, H., Zipprich, J.T., Svitkin, Y.V., Rivas, F., Jinek, M., Wohlschlegel, J., Doudna, J.A., Chen, C.-Y.A., Shyu, A.-B., Yates, J.R., Hannon, G.J., Filipowicz, W., Duchaine, T.F. and Sonenberg, N. (2009) 'Mammalian miRNA RISC Recruits CAF1 and PABP to Affect PABP-Dependent Deadenylation', Molecular cell, 35(6), pp. 868-880.