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Ars2 Links the Nuclear Cap-Binding Complex to RNA Interference and Cell Proliferation

Joshua J. Gruber, D. Steven Zatechka, Leah R. Sabin, Jeongsik Yong, Julian J. Lum, Mei Kong, Wei-Xing Zong, Zhenxi Zhang, Chi-Kong Lau, Jason Rawlings, Sara Cherry, James N. Ihle, Gideon Dreyfuss and Craig B. Thompson

Cell 138 (2): 328-339, July 24, 2009.
doi:10.1016/j.cell.2009.04.046

This week’s long summary and analysis by David Garcia:

Synopsis:
The authors demonstrate a clear role for the mammalian Ars2 protein in cell proliferation, as well as an interaction with the Cap-Binding Complex. Depletion of Ars2 reduces the miRNA or siRNA directed repression of two reporter constructs, and levels of mature miRNAs for two out of four miRNAs investigated. Ars2 also co-precipitates with Drosha, but not Dicer, and pre- or mature let-7 can rescue some or all of the loss of repression of the reporters associated with Ars2 depletion. The authors are less successful at directly connecting Ars2’s effect on RNAi that they observed to a potential role in primary-miRNA to pre-miRNA processing by Drosha. The processing assays should have tested more substrates, and their variability is not explained clearly enough.

Detail:
This paper aims to demonstrate a critical role for the mammalian protein Ars2 in cell proliferation, and associate this role with other evidence that it also affects the stability or processing efficiency of a couple of primary miRNA transcripts. While initially setting out to study how mammalian Ars2 imparts a resistance to arsenic oxide treatment, the authors discovered previous studies had focused on a partial clone of the protein. In this study, the full-length protein affects arsenic treatment in a manner opposite to what had been demonstrated before; its reduction has a profound influence on cell proliferation; and it contains a number of domains common to RNA binding proteins, and homology to the Arabidopsis protein SERRATE known to affect the processing of primary miRNA transcripts by Dicer-Like 1 (the plant Drosha).

The paper starts by showing that when Ars2 is knocked down using shRNAs, 3T3 cells die more slowly compared to controls when treated with arsenic oxide (Fig1B). Also observed when depleting Ars2 is a cell proliferation defect, using colony formation assays and counting population doublings (Fig1D,F). To investigate further, the authors generate a floxed allele of Ars2 in mESCs, and then generate mice from which they derive immortalized MEFs that they can infect with Cre expressing retrovirus. These Ars2 depleted cells exhibit the same proliferation defects as had been seen with the cell lines (Fig2B). Examination of various tissues from the Ars2 depleted adult mice show that in tissues that have relatively high cell proliferation and normally high expression levels of Ars2, like hematopoietic tissues, there is decreased cellularity or increased apoptosis (Fig2D). In contrast, there are no such differences observed between the transgenic and WT mice in other lower proliferating tissues.

To find out what other proteins Ars2 interacts with, a flagged-Ars2 is expressed, and co-precipitates with several components of the cap-binding complex (CBC), including CBP80, CBP20, and importin-alpha (Fig3A). Ars2 and the CBC interact with 7-Methyl-Guanosine capped RNAs (Fig4A), as would be expected for a component of the CBC. In addition, Ars2 shuttles between the nucleus and cytoplasm (Fig4B), like other CBC components.

Given SERRATE’s known role in miRNA biogenesis in Arabidopsis, the authors address whether the mammalian Ars2 in involved in RNAi. Two types of luciferase reporters are created: one with 3X let-7 (7mer) miRNA binding sites derived from the C. elegans lin-28 3 prime UTR; and another with a perfectly matched site for the let-7 miRNA (~22mer match). In HeLa cells, they first transfect siRNAs to knock down Ars2 or CBP80 or Ago2 (positive control), and then transfect the reporters to see how repression compares to cells treated with a control siRNA. The siRNAs against Ars2 and CBP80 inhibit let-7 directed repression at levels comparable to knockdown of Ago2 for both reporters, with a more pronounced effect on the miRNA sites reporter (Fig5A).

I am surprised knocking down Ago2 only affected repression ~2.5 fold for the perfect site reporter, and not much greater. Let-7 levels are very high in HeLa cells. However, there could be some complications of knocking down Ago2 with an siRNA. Unfortunately, while the authors state a reporter with a mutated let-7 seed site led to a loss of repression under all conditions, they do not show how this loss of repression compares to the loss they observed from Ars2, CBP80, and Ago2 siRNAs. This would have been a useful control to show.

Next the authors show that addition of excess let-7 duplex can rescue loss of Ars2, but not Ago2, for both reporters (Fig5B), suggesting Ars2 may affect some step in miRNA biogenesis before Dicer. Depletion of Ars2 or DGCR8 with two different siRNAs each led to a decrease in mature let-7 levels (~50% reduction)(Fig5C,D), and the same story for mature miR-21 (expressed very highly in HeLa). While they couldn’t detect pre-let-7 by Northern (I’m surprised by this), they did see a reduction in pre-miR-21 in Ars2 depleted HeLa cells. The reduction in pre or mature was not observed for all miRNAs they probed: there was no change for miR-30a or miR-16 (FigS3).

To investigate which step of miRNA processing is affected by Ars2 depletion, they immunoprecipitate a tagged Drosha and Dicer from 293T cells. Ars2 and CBP80 both co-precipitate with Drosha but not Dicer, and this interaction is not RNA dependent (Fig6A). Addition of excess pre-let-7 rescued some of the loss of repression from Ars2 or CBP80 depleted cells (but not Ago2 depleted)(Fig6C). Depletion of Ars2 also led to a decrease in pri-miR-21 levels by qRT-PCR, indicating that Ars2 may also influence the stability of Drosha substrates (Fig6D).

A pri-miRNA processing assay is employed to address the role of Ars2 in affecting the fidelity of Drosha mediated processing. In these assays, they compare the processing of an in vitro transcribed pri-miR-155 between different cell extracts. The authors state that there is an observed reduction in pri to pre processing in the MEF Ars2 KO cells compared to WT (Fig6F). However, it may be problematic to compare two different cell extracts that are almost guaranteed to differ in more than just Ars2 expression. In the Ars2 KO, the “correct” pre band mostly disappears, but a slightly smaller band also present in the WT intensifies, without explanation. They only show data for a single pri-miRNA, 155 (but say comparable results were obtained for miR-21). They quantitate the reduction in “correct” processing as 3-fold for miR-155 (Fig6G).

The paper ends by weakly connecting the observed effect of Ars2 on cell proliferation with its potential effect on miRNA processing. The authors first re-visit the proliferation point by showing that serum starved MEFs that exit the cell cycle lose Ars2 expression (Fig7A). They confirm Ars2’s proliferation dynamic expression in the hematopoietic cell line Bax-/- Bax-/- (Fig7C). A primary miRNA processing assay, comparing processing of pri-miR-155 (in vitro transcribed as before) between 10% serum grown or 0.1% serum starved MEF extracts, is claimed to show a decrease in pri to pre in the Ars2 depleted serum starved extracts (Fig 7D). While there seems to be an increase in heterogeneity in pre, the total reduction in pre in the serum starved cells is not robust. As before, the implication that we are to assume that variability between different cell extracts, from different cell lines, or grown under vastly different conditions, cannot contribute to variability in pri to pre processing is, I think, not well founded. Moreover, the authors only show results from the testing of one miRNA, miR-155. There is insufficient explanation for bands nearby those they deem as “correctly processed.” While the authors have shown clearly how Ars2 affects cell proliferation, interacts with the CBC, and interacts with Drosha, I don’t believe they have clearly explained how Ars2 affects RNAi with their proposed mechanism of affecting miRNA biogenesis. Hopefully there will be stronger data to support this hypothesis in future papers.