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Versatility of MicroRNA biogenesis.

Volk N, Shomron N - PLoS ONE (2011)

Bottom Line: Moreover, over-expression of several factors involved in miRNA biogenesis, including Exportin-5, Drosha, NF90a, NF45 and KSRP, displayed bidirectional effects on pre/mature miRNA ratios, suggesting their intricate biogenesis sensitivity.Knock-down or over-expression of these genes suggested that hnRNPR inhibits, whereas hnRNPH1 facilitates, miRNA processing.Overall, our results emphasize that miRNA biogenesis is versatile.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

ABSTRACT
MicroRNAs (miRNAs) are short single-stranded RNA molecules that regulate gene expression. MiRNAs originate from large primary (pri) and precursor (pre) transcripts that undergo various processing steps along their biogenesis pathway till they reach their mature and functional form. It is not clear, however, whether all miRNAs are processed similarly. Here we show that the ratio between pre-miRNA and mature miRNA forms varies between different miRNAs. Moreover, over-expression of several factors involved in miRNA biogenesis, including Exportin-5, Drosha, NF90a, NF45 and KSRP, displayed bidirectional effects on pre/mature miRNA ratios, suggesting their intricate biogenesis sensitivity. In an attempt to identify additional factors that might explain the versatility in miRNA biogenesis we have analyzed the contribution of two hnRNP family members, hnRNPH1 and hnRNPR. Knock-down or over-expression of these genes suggested that hnRNPR inhibits, whereas hnRNPH1 facilitates, miRNA processing. Overall, our results emphasize that miRNA biogenesis is versatile.

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Expression levels of factors involved in miRNA biogenesis.Western blot analysis using (A) anti-myc antibody or (B) anti-Flag antibody of HeLa cells over-expressing either (A) Exportin-5, (B) Drosha, KSRP or NF-90a and NF-45.
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pone-0019391-g001: Expression levels of factors involved in miRNA biogenesis.Western blot analysis using (A) anti-myc antibody or (B) anti-Flag antibody of HeLa cells over-expressing either (A) Exportin-5, (B) Drosha, KSRP or NF-90a and NF-45.

Mentions: In order to test whether any of the miRNA biogenesis factors are accountable for the observed differential levels of pre- and mature miRNA forms we over-expressed the following known miRNA biogenesis pathway genes: (i) Drosha, which cleaves the pri-miRNA into the pre-miRNA form of all but very few exceptional miRNAs; (ii) Nuclear factors NF90a and NF45, which inhibit the cleavage of pri-miRNA to pre-miRNA (shown for pri-let-7a and pri-miR-21) [5]; (iii) KSRP, which binds the terminal loop of the precursor and enhances its maturation (shown for miR-1, miR-15, miR-16, miR-20a, miR-21, miR-26b, miR-27b, miR-106a, miR-125, miR-196, miR-206 and let-7 on the basis of its high-affinity binding to their terminal loop) [6]; (iv) Exportin-5, which exports the pre-miRNA from the nucleus to the cytoplasm [3]. After allowing high levels of these proteins to accumulate in the cells (Fig. 1; 48 hr after transfection), RNA was extracted and the levels of pre- and mature miRNAs were quantified relative to control plasmid transfection.


Versatility of MicroRNA biogenesis.

Volk N, Shomron N - PLoS ONE (2011)

Expression levels of factors involved in miRNA biogenesis.Western blot analysis using (A) anti-myc antibody or (B) anti-Flag antibody of HeLa cells over-expressing either (A) Exportin-5, (B) Drosha, KSRP or NF-90a and NF-45.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3091858&req=5

pone-0019391-g001: Expression levels of factors involved in miRNA biogenesis.Western blot analysis using (A) anti-myc antibody or (B) anti-Flag antibody of HeLa cells over-expressing either (A) Exportin-5, (B) Drosha, KSRP or NF-90a and NF-45.
Mentions: In order to test whether any of the miRNA biogenesis factors are accountable for the observed differential levels of pre- and mature miRNA forms we over-expressed the following known miRNA biogenesis pathway genes: (i) Drosha, which cleaves the pri-miRNA into the pre-miRNA form of all but very few exceptional miRNAs; (ii) Nuclear factors NF90a and NF45, which inhibit the cleavage of pri-miRNA to pre-miRNA (shown for pri-let-7a and pri-miR-21) [5]; (iii) KSRP, which binds the terminal loop of the precursor and enhances its maturation (shown for miR-1, miR-15, miR-16, miR-20a, miR-21, miR-26b, miR-27b, miR-106a, miR-125, miR-196, miR-206 and let-7 on the basis of its high-affinity binding to their terminal loop) [6]; (iv) Exportin-5, which exports the pre-miRNA from the nucleus to the cytoplasm [3]. After allowing high levels of these proteins to accumulate in the cells (Fig. 1; 48 hr after transfection), RNA was extracted and the levels of pre- and mature miRNAs were quantified relative to control plasmid transfection.

Bottom Line: Moreover, over-expression of several factors involved in miRNA biogenesis, including Exportin-5, Drosha, NF90a, NF45 and KSRP, displayed bidirectional effects on pre/mature miRNA ratios, suggesting their intricate biogenesis sensitivity.Knock-down or over-expression of these genes suggested that hnRNPR inhibits, whereas hnRNPH1 facilitates, miRNA processing.Overall, our results emphasize that miRNA biogenesis is versatile.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

ABSTRACT
MicroRNAs (miRNAs) are short single-stranded RNA molecules that regulate gene expression. MiRNAs originate from large primary (pri) and precursor (pre) transcripts that undergo various processing steps along their biogenesis pathway till they reach their mature and functional form. It is not clear, however, whether all miRNAs are processed similarly. Here we show that the ratio between pre-miRNA and mature miRNA forms varies between different miRNAs. Moreover, over-expression of several factors involved in miRNA biogenesis, including Exportin-5, Drosha, NF90a, NF45 and KSRP, displayed bidirectional effects on pre/mature miRNA ratios, suggesting their intricate biogenesis sensitivity. In an attempt to identify additional factors that might explain the versatility in miRNA biogenesis we have analyzed the contribution of two hnRNP family members, hnRNPH1 and hnRNPR. Knock-down or over-expression of these genes suggested that hnRNPR inhibits, whereas hnRNPH1 facilitates, miRNA processing. Overall, our results emphasize that miRNA biogenesis is versatile.

Show MeSH