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Post-transcriptional Gene Silencing Goes Nuclear

June 2018 

Recently, researchers in France identified a novel protein that controls post-transcriptional gene silencing. Their finding is significant because it is the first-time post-transcriptional silencing has included nucleoplasmic mechanisms.

The researchers stumbled upon this finding while trying to understand how microRNA (miRNA) targets and modulates RNA degradation.

Traditional methods to identify miRNA binding sites have been shown to produce approximately 70% false or negative targets, so Bottini and colleagues decided to circumvent this problem by identifying RNA binding proteins that bind to messenger RNAs (mRNAs) to control miRNA targeting.

"In support of this mechanism, it has been shown that some RNA-binding proteins associate with specific mRNAs and interfere with specific miRNA-binding sites to either inhibit or enhance miRNA targeting. This result leads to the concept of a sequence microenvironment surrounding miRNA-binding sites that plays an important role in regulating miRNA activity," the authors explain.

To explore this, the researchers used a quantitative proteomic analysis approach to identify proteins that interact with Argonaute (Ago) proteins, mainly Ago2, to form the miRNA-induced silencing complex (miRISC).

HITS-CLIP
Figure 1: HITS-CLIP. High-throughput methods for identification of miRNA target genes. HITS-CLIP utilizes ultraviolet (UV)-induced covalent crosslinking to stabilize RNA-Argonaute (AGO) protein complexes in miRISC, thereby enhancing the ability to capture more transient miRNA-mRNA interactions, prior to immunoprecipitation (IP) with antibodies. Massively parallel sequencing (MPS) of bound RNAs then allows comprehensive identification of functional miRNA-target mRNA interaction sites. MicroRNA in Aging: From Discovery to Biology. Available from: https://www.researchgate.net/publication/236600524_MicroRNA_in_Aging_From_Discovery_to_Biology [accessed Jun 03 2018]. Courtesy of Jung HJ and Suh Y, 2012. Material used through CC 2.5.

After they identified splicing factor proline/glutamine-rich protein (Sfpq) as a protein that interacts with nucleoplasmic miRISC in different human and mouse cell lines, they hypothesized that Sfpq could modulate miRNA-binding activity, and ultimately control gene expression.

To understand which miRNAs are modulated by Sfpq binding, they then used High-Throughput Sequencing of RNA isolated by CrossLinking IP (HITS-CLIP) (see Figure 1) in the presence or absence of Sfpq to find interactors. Notably, the authors used the TriLink CleanTag® Small RNA Library Prep Kit, which blocks adapter dimer formation, increasing mappable sequencing reads.

Ultimately, Bottini and colleagues found that Sfpq promotes miRNA binding for a subset of miRNA, including oncogene Lin28, and controls mRNA silencing at specific miRNA-binding sites.

They also noted that Sfpq aggregates on target 3 UTRs to promote miRNA targeting, leading them to hypothesize that theses aggregates modulate the recruitment functions of miRISC by changing the secondary structure of the target 3UTRs.

A model for the Sfpq-dependent control of miRNA targeting
Figure 2: A model for the Sfpq-dependent control of miRNA targeting. Courtesy of Bottini et al. 2017. Material used through CC 4.0.

These data together present a novel mechanism for post-transcription gene regulation that occurs in the nucleus, expanding the traditional view of gene regulation (see Figure 2).

“Overall, these data indicate that the presence of specific Sfpq-binding sites determines the fate of a cohort of mRNAs, where Sfpq forms long aggregates in the 3 UTR to modulate 3 UTR folding for the proper positioning/recruitment of miRNAs to selected binding sites, whereas avoiding random binding of miRNAs that would not be effective”, the authors conclude.

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