This is an essay reviewing the research published in PNAS in December 15, 2015. To read the research paper in prior to reading the essay, please refer to the link below: Casein kinase II promotes target silencing by miRISC through direct phosphorylation of the DEAD-box RNA helicase CGH-1 https://www.ncbi.nlm.nih.gov/pubmed/26669440
While miRNA has been discovered as a key regulator that intervenes in various posttranscriptional protein regulation, specific mechanisms that lead to miRISC activity are yet to be fully understood. This research provided experimental evidence to explain the role of Casein kinase II(CK2), a prosurvival kinase that regulates significant mechanism, in miRISC binding to mRNA targets in C. elegans. Their step-by-step approach first illustrates that the downregulation of two genes encoding subunits of CK2, kin-3 and kin-10, compromises the miRISC activity, and concludes that CK2 promotes miRISC function. Further experiments are done to demonstrate that CGH-1, a potential miRISC-target interaction facilitator, should be phosphorylated by CK2 to function in miRNA pathways. Suggesting some possible specific mechanisms by which miRISC target binding is regulated by phosphorylation of CGH-2 by CK2, the research illustrates CK2 activity that can be further studied to identify the regulation of the miRNA pathway.
Linking Function of CK2 and miRNA Target Silencing Indirectly Based on RNAi of Subunits of CK2
In the first part (until half of the fourth page) of the research, various experiments show that RNAi of CK2 subunits phenocopies loss of miRISC components. First, when subjected to CK2 RNAi, similar defects as RNAi of the miRISC component is shown in adult hypodermal remodeling. Second, adult lethargus due to mutations of let-7 miRNA family shows compatible levels of that caused by CK2 RNAi. CK2 RNAi also enhances the mutant phenotype of non-let-7 family miRNA, lsy-6 and miR-35. Finally, the miRNA Target Silencing of lin-41, daf-12, lin-14, mef-2 are suppressed when treated with CK2 RNAi.
The Logical Fallacy in the Relationship
In these experiments, depleting either kin-3 or kin-10 by RNAi – specific, relatively easy, although does not silence completely – is an adequate way to monitor the activity of CK2 without hindering embryonic development, as CK2 is significant in diverse cell mechanism, including proliferation in germ line . The subunits were found to physically interact with AIN-1, a homolog of GW182 which is essential in miRNA mediated gene silencing. Moreover, the use of a sensitized miRNA environment was an adequate method, as it undermines the redundancy of miRNA function. However, that depleting CK2 leads to similar defects as RNAi of the miRISC component is not logically equivalent to that CK2 promotes miRISC function. Therefore, some additional efforts are done to clarify the relation between CK2 and miRISC and avoid logical fallacy. The research verifies that kin-10 genetic mutants, as well as kin-10 RNAi, lead to increased Rup phenotype and that CK2 depletion does not lead to enhanced embryonic lethality in wild-type animals (of both mir-48 and mir-35-41). The latter result confirms that CK2 interferes with embryonic lethality by affecting the miRISC pathway, making the conclusion that CK2 facilitates miRISC function plausible
Understanding Specific Mechanism by which CK2 Promotes Target Silencing
With this background, further experiments are done to locate the CK2 function in the miRNA pathway. Above all, the research rejects the possibility of CK2 affecting the stability of mature miRNA, precursor level of miRNA, and the stability of core miRISC proteins based on each result of deep sequencing (Fig.3A), Northern blot (Fig. 3B), and Western blot (Fig.3C). Rather, CK2 is expected to cause downstream regulation of miRNA; the result of RNA immunoprecipitation (RIP) with GFP::ALG-1 shows that while the association of miRNA to miRISC is unaffected, the association of target mRNAs decreased despite the general up-regulation in level when treated with kin-3 or kin-10 RNAi. Moreover, the research finds out based on mass spectroscopy data of immunopurified CGH-1 that CGH-1 is phosphorylated in a CK2-dependent manner. CGH-1 of kin-3 RNAi sample contain non-phosphorylated S2 site whereas that of empty vector RNAi sample do. Various CGH-1 phosphorylation mutants (S2 variants) show defective miRNA-dependent CGH-1 function in Pvul and retarded alae phenotype. However, during postdauer development, other factors are thought to interfere in the CK2 mechanism that promotes miRISC target silencing since the postdauer experiment results do not abide by the overall conclusion that CK2 phosphorylation of CGH-1 is required for miRISC function.
Do not Accept the Result Without Examining Once More
In this latter part of the research in vitro kinase assay and GST-purification are used to detect the specific location of CK2 activity. However, in Figure 4D that presented SDS PAGE, the result of 32P autoradiogram that indicates the site 1 as phosphorylation site of CK2 does not comply with the result of Coomassie blue stained gel that rather presents the site 3 or 4 as a better site of phosphorylation than site 1. SDS PAGE is an appropriate method to separate GST-tagged CGH-1 peptides by size, and Coomassie blue staining is an appropriate way to detect proteins of size 0.1–0.5 μg. Therefore, the Coomassie blue staining result should be scrutinized further on validity before drawing the conclusion.
Another thing that needs caution when interpreting the result is the expression level of protein and influence of tag. In fact, the phosphodefective lines that are expressed substantially lower showed a significantly higher rate of Pvul, undermining the discrepancy of expression levels of cgh-1::gfp between the phosphorylation mutants and wild-type. Yet, there is a possibility of steric hindrance due to the large size of GFP and GST tag – GFP tag is ~240aa , GST tag is about 220aa while CGH-1 is 430aa – which must be examined to verify the result.
Other than this, some results of the experiments manifested through the diagrams are dubious. In Figure 2C, the change of the thickness of the band in LIN-14 of kin-10(RNAi) and alg-1(RNAi) shows dubious alteration. Moreover, if the initial level LIN-14 before the treatment was provided, the silencing of lin-4 target would have been better compared. The data value of mef-2 reporter quantification (Fig.2E) and relative miRNA levels of kin-3(RNAi) sample (Fig.3E) shows a large deviation, making the accuracy and reliability doubtful.
Despite these flaws, this research provides coherent evidence that CK2 facilitates miRISC function by phosphorylating CGH-1 in the S2 site during the continuous development of Caenorhabditis elegans. The overall step-by-step approach to draw the conclusion and some additional experiments to eliminate logical fallacies are notable. In the end, the research further develops speculation on how the phosphorylation of CGH-1 leads to promoting target silencing by miRISC – recruitment of cofactors for miRISC RNP granules may be facilitated by the phosphorylated CGH-1.
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