Are Argonaute-Associated Tiny RNAs Junk, Inferior miRNAs, or a New Type of Functional RNAs?

The biosynthesis pathways of microRNAs (miRNAs) have been well characterized with the identification of the required components. miRNAs are synthesized from the transcripts of miRNA genes and other RNAs, such as introns, transfer RNAs, ribosomal RNAs, small nucleolar RNAs, and even viral miRNAs. These small RNAs are loaded into Argonaute (AGO) proteins and recruit the effector complexes to target mRNAs, repressing their gene expression post-transcriptionally. While mature miRNAs were defined as 19–23 nucleotides (nt), tiny RNAs (tyRNAs) shorter than 19 nt have been found to bind AGOs as equivalent or lesser miRNAs compared to their full-length mature miRNAs. In contrast, my recent study revealed that when human AGO3 loads 14 nt cleavage-inducing tyRNAs (cityRNAs), comprised of the first 14 nt of their corresponding mature miRNA, it can become a comparable slicer to AGO2. This observation raises the possibility that tyRNAs play distinct roles from their mature form. This minireview focuses on human AGO-associated tyRNAs shorter than 19 nt and discusses their possible biosynthesis pathways and physiological benefits, including how tyRNAs could avoid target-directed miRNA degradation accompanied by AGO polyubiquitination.

Widespread microRNA degradation elements in target mRNAs can assist the encoded proteins

Binding of microRNAs (miRNAs) to mRNAs normally results in post-transcriptional repression of gene expression. However, extensive base-pairing between miRNAs and target RNAs can trigger miRNA degradation, a phenomenon called target RNA-directed miRNA degradation (TDMD). Here, we systematically analyzed Argonaute-CLASH (cross-linking, ligation, and sequencing of miRNA–target RNA hybrids) data and identified numerous candidate TDMD triggers, focusing on their ability to induce nontemplated nucleotide addition at the miRNA 3′ end. When exogenously expressed in various cell lines, eight triggers induce degradation of corresponding miRNAs. Both the TDMD base-pairing and surrounding sequences are essential for TDMD. CRISPR knockout of endogenous trigger or ZSWIM8, a ubiquitin ligase essential for TDMD, reduced miRNA degradation. Furthermore, degradation of miR-221 and miR-222 by a trigger in BCL2L11, which encodes a proapoptotic protein, enhances apoptosis. Therefore, we uncovered widespread TDMD triggers in target RNAs and demonstrated an example that could functionally cooperate with the encoded protein.

scanMiR: a biochemically-based toolkit for versatile and efficient microRNA target prediction

Despite the importance of microRNAs (miRNAs) in regulating a broad variety of biological processes, accurately predicting the transcripts they repress remains a challenge. Recent research suggests improved miRNA target prediction using a biochemical model combined with empirically-derived affinity predictions across 12mer sequences. Here, we translate this approach into a generally applicable, flexible and user-friendly tool (scanMiR). By compressing and handling miRNA 12mer affinity predictions into lightweight models, scanMiR can efficiently scan for both canonical and non-canonical binding sites on transcripts and custom sequences (including circRNAs and lncRNAs). Aggregation of binding sites into predicted transcript repression using a generalized biochemical model correlates better with experimental data than the most accurate alternative publicly available predictions. Moreover, a flexible 3'-supplementary alignment enables scanMiR to highlight and visualize unconventional modes of miRNA target mRNA interactions, such as bindings leading to target-directed miRNA degradation (TDMD) and slicing. By specifically scanning for these unconventional binding sites in brain-derived expression data, we provide a first systematic overview of potential TDMD and slicing sites on brain-specific lncRNAs as well as circRNAs. Finally, in addition to the main bioconductor package implementing these functions, we provide a user-friendly web application enabling the scanning of sequences, the visualization of predicted bindings, and the browsing of predicted target repression.

The ZSWIM8 ubiquitin ligase mediates target-directed microRNA degradation

MicroRNAs (miRNAs) associate with Argonaute (AGO) proteins to direct widespread post-transcriptional gene repression. Although association with AGO typically protects miRNAs from nucleases, extensive pairing to some unusual target RNAs can trigger miRNA degradation. Here we found that this target-directed miRNA degradation (TDMD) required the ZSWIM8 Cullin-RING E3 ubiquitin ligase. This and other findings suggested and supported a mechanistic model of TDMD in which target-directed proteolysis of AGO by the ubiquitin–proteasome pathway exposes the miRNA for degradation. Moreover, loss-of-function studies indicated that the ZSWIM8 Cullin-RING ligase accelerates degradation of numerous miRNAs in cells of mammals, flies, and nematodes, thereby specifying the half-lives of most short-lived miRNAs. These results elucidate the mechanism of TDMD and expand its inferred role in shaping miRNA levels in bilaterian animals.

miRNA degradation in the mammalian brain

MicroRNAs (miRNAs) are short, noncoding RNAs that are evolutionarily conserved across many different species. miRNA regulation of gene expression, specifically in the context of the mammalian brain, has been well characterized; however, the regulation of miRNA degradation is still a focus of ongoing research. This review focuses on recent findings concerning the cellular mechanisms that govern miRNA degradation, with an emphasis on target-mediated miRNA degradation and how this phenomenon is uniquely poised to maintain homeostasis in neuronal systems.

Impaired miRNA degradation by post-transcriptional addition of 3’ cytosine and adenine in T cell activation

ABSTRACTMiRNA repertoire of T cells undergoes extensive changes in response to activation. Whereas global miRNA downregulation occurs few hours after activation, some individual miRNAs are specifically up- or down-regulated. In this study, we have assessed miRNA expression and post-transcriptional modification kinetics in human primary CD4+ T cells upon short-term stimulation with αCD3αCD28 or IFN I using Next Generation Sequencing. Multiple miRNAs not related before with T cell activation profile have been identified as differentially expressed. Downregulated miRNAs presented higher 3’ uridylation. Dis3L2 and Eri1 (3’ to 5’ exoribonucleases that prefer uridylated RNA as substrates) increased their expression upon TCR stimulation, probably generating an adverse environment for miRNAs. Remarkably, non-templated cytosine additions to 3’ end, previously unknown to be a relevant post-transcriptional modification mechanism, were overrepresented in upregulated miRNAs, together with high levels of adenylation. In the midst of an increasing presence of exoribonucleases, miRNAs multiplying their levels may successfully escape degradation due to 3’ cytosine and adenine addition. These protective signals open a new avenue to improve miRNA stability for therapy in T cells.

SERRATE interacts with the nuclear exosome targeting (NEXT) complex to degrade primary miRNA precursors in Arabidopsis

SERRATE/ARS2 is a conserved RNA effector protein involved in transcription, processing and export of different types of RNAs. In Arabidopsis, the best-studied function of SERRATE (SE) is to promote miRNA processing. Here, we report that SE interacts with the nuclear exosome targeting (NEXT) complex, comprising the RNA helicase HEN2, the RNA binding protein RBM7 and one of the two zinc-knuckle proteins ZCCHC8A/ZCCHC8B. The identification of common targets of SE and HEN2 by RNA-seq supports the idea that SE cooperates with NEXT for RNA surveillance by the nuclear exosome. Among the RNA targets accumulating in absence of SE or NEXT are miRNA precursors. Loss of NEXT components results in the accumulation of pri-miRNAs without affecting levels of miRNAs, indicating that NEXT is, unlike SE, not required for miRNA processing. As compared to se-2, se-2 hen2-2 double mutants showed increased accumulation of pri-miRNAs, but partially restored levels of mature miRNAs and attenuated developmental defects. We propose that the slow degradation of pri-miRNAs caused by loss of HEN2 compensates for the poor miRNA processing efficiency in se-2 mutants, and that SE regulates miRNA biogenesis through its double contribution in promoting miRNA processing but also pri-miRNA degradation through the recruitment of the NEXT complex.

SERRATE interacts with the nuclear exosome targeting (NEXT) complex to degrade primary miRNA precursors in Arabidopsis

SERRATE/ARS2 is a conserved RNA effector protein involved in transcription, processing and export of different types of RNAs. In Arabidopsis, the best-studied function of SERRATE (SE) is to promote miRNA processing. Here, we report that SE interacts with the Nuclear Exosome Targeting (NEXT) complex, comprising the RNA helicase HEN2, the RNA binding protein RBM7 and one of the two zinc-knuckle proteins ZCCHC8A/ZCCHC8B. The identification of common targets of SE and HEN2 by RNA-seq supports the idea that SE cooperates with NEXT for RNA surveillance by the nuclear exosome. Among the RNA targets accumulating in absence of SE or NEXT are miRNA precursors. Loss of NEXT components results in the accumulation of pri-miRNAs without affecting levels of miRNAs, indicating that NEXT is, unlike SE, not required for miRNA processing. As compared to se-2, se-2 hen2-2 double mutants showed increased accumulation of pri-miRNAs, but partially restored levels of mature miRNAs and attenuated developmental defects. We propose that the slow degradation of pri-miRNAs caused by loss of HEN2 compensates for the poor miRNA processing efficiency in se-2 mutants, and that SE regulates miRNA biogenesis through its double contribution in promoting miRNA processing but also pri-miRNA degradation through the recruitment of the NEXT complex.

Lentivirus-mediated long-term overexpression of specific microRNA for complementary miRNA pairs in mammalian cells

The establishment of a method that would overexpress or suppress of specific microRNA activity is essential for the functional analysis of these molecules and for the development of miRNA therapeutic applications. There already exist excellent ways to inhibit miRNA function in vitro and in vivo by overexpressing miRNA target sequences, which include miRNA ‘decoys’, ‘sponges’, or ‘antagomirs’ that are complementary to an miRNA seed region. Conversely, no methods to induce stable gain-of-function phenotypes for specific miRNAs have, as yet, been reported. Furthermore, the discovery of complementary miRNA pairs raises suspicion regarding the existing methods used for miRNA overexpression. In our study, we will study whether the traditional methods for miRNA overexpression can be used for specific miRNA overexpression while complementary miRNA pairs exist. In addition, we test various miRNA-expression cassettes that were designed to efficiently overexpress specific miRNA through the shRNA lentivirus expression system. We report the optimal conditions that were established for the design of such miRNA-expression cassettes. We finally demonstrate that the miRNA-expression cassettes achieve efficient and long-term overexpression of specific miRNAs. Meanwhile, our results also support the notion that miRNA–miRNA interactions are implicated in potential, mutual regulatory patterns and beyond the seed sequence of miRNA, extensive pairing interactions between a miRNA and its target also lead to target-directed miRNA degradation. Our results indicate that our method offers a simple and efficient means to over-express the specific miRNA with long-term which will be very useful for future studies in miRNA biology, as well as contributed to the development of miRNA-based therapy for clinical applications.