Canonical microRNAs Research Articles

Characterization of Viral and Human RNAs Smaller than Canonical MicroRNAs

Recently identified small (20 to 40 bases) RNAs, such as microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) participate in important cellular pathways. In this report, we systematically characterized several novel features of human and viral RNA products smaller than miRNAs. We found that Kaposi sarcoma-associated herpesvirus K12-1 miRNA (23 bases) associates with a distinct, unusually small (17-base) RNA (usRNA) that can effectively downregulate a K12-1 miRNA target, human RAD21, suggesting that stable degradation-like products may also contribute to gene regulation. High-throughput sequencing reveals a diverse set of human miRNA-derived usRNAs and other non-miRNA-derived usRNAs. Human miRNA-derived usRNAs preferentially match to 5' ends of miRNAs and are also more likely to associate with the siRNA effector protein Ago2 than with Ago1. Many non-miRNA-derived usRNAs associate with Ago proteins and also frequently contain C-rich 3'-specific motifs that are overrepresented in comparison to Piwi-interacting RNAs and transcription start site-associated RNAs. We postulate that approximately 30% of usRNAs could have evolved to participate in biological processes, including gene silencing.

The non-coding RNA of the multidrug resistance-linked vault particle encodes multiple regulatory small RNAs

Vault particles are conserved organelles implicated in multidrug resistance and intracellular transport. They contain three different proteins and non-coding vault RNAs (vRNAs). Here we show that human vRNAs produce several small RNAs (svRNAs) by mechanisms different from those in the canonical microRNA (miRNA) pathway. At least one of these svRNAs, svRNAb, associates with Argonaute proteins to guide sequence-specific cleavage and regulate gene expression similarly to miRNAs. We demonstrate that svRNAb downregulates CYP3A4, a key enzyme in drug metabolism. Our findings expand the repertoire of small regulatory RNAs and assign, for the first time, a function to vRNAs that may help explain the association between vault particles and drug resistance.

Genome-Wide and Organ-Specific Landscapes of Epigenetic Modifications and Their Relationships to mRNA and Small RNA Transcriptomes in Maize

Maize (Zea mays) has an exceptionally complex genome with a rich history in both epigenetics and evolution. We report genomic landscapes of representative epigenetic modifications and their relationships to mRNA and small RNA (smRNA) transcriptomes in maize shoots and roots. The epigenetic patterns differed dramatically between genes and transposable elements, and two repressive marks (H3K27me3 and DNA methylation) were usually mutually exclusive. We found an organ-specific distribution of canonical microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs), indicative of their tissue-specific biogenesis. Furthermore, we observed that a decreasing level of mop1 led to a concomitant decrease of 24-nucleotide siRNAs relative to 21-nucleotide miRNAs in a tissue-specific manner. A group of 22-nucleotide siRNAs may originate from long-hairpin double-stranded RNAs and preferentially target gene-coding regions. Additionally, a class of miRNA-like smRNAs, whose putative precursors can form short hairpins, potentially targets genes in trans. In summary, our data provide a critical analysis of the maize epigenome and its relationships to mRNA and smRNA transcriptomes.

Embryonic stem cell–specific microRNAs regulate the G1-S transition and promote rapid proliferation

Dgcr8 knockout embryonic stem (ES) cells lack microprocessor activity and hence all canonical microRNAs (miRNAs). These cells proliferate slowly and accumulate in G1 phase of the cell cycle1. Here, by screening a comprehensive library of individual miRNAs in the background of the Dgcr8 knockout ES cells, we report that multiple ES cell-specific miRNAs, members of the miR-290 family, rescue the ES cell proliferation defect. Furthermore, rescued cells no longer accumulate in the G1 phase of the cell cycle. These miRNAs function by suppressing several key regulators of the G1/S transition. These results show that post-transcriptional regulation by miRNAs promotes the G1/S transition of the ES cell cycle enabling their rapid proliferation. Furthermore, our screening strategy provides an alternative and powerful approach for uncovering the role of individual miRNAs in biological processes as it overcomes the common problem of redundancy and saturation in the miRNA system.

Mouse ES cells express endogenous shRNAs, siRNAs, and other Microprocessor-independent, Dicer-dependent small RNAs

Canonical microRNAs (miRNAs) require two processing steps: the first by the Microprocessor, a complex of DGCR8 and Drosha, and the second by a complex of TRBP and Dicer. dgcr8Delta/Delta mouse embryonic stem cells (mESCs) have less severe phenotypes than dicer1Delta/Delta mESCs, suggesting a physiological role for Microprocessor-independent, Dicer-dependent small RNAs. To identify these small RNAs with unusual biogenesis, we performed high-throughput sequencing from wild-type, dgcr8Delta/Delta, and dicer1Delta/Delta mESCs. Several of the resulting DGCR8-independent, Dicer-dependent RNAs were noncanonical miRNAs. These derived from mirtrons and a newly identified subclass of miRNA precursors, which appears to be the endogenous counterpart of shRNAs. Our analyses also revealed endogenous siRNAs resulting from Dicer cleavage of long hairpins, the vast majority of which originated from one genomic locus with tandem, inverted short interspersed nuclear elements (SINEs). Our results extend the known diversity of mammalian small RNA-generating pathways and show that mammalian siRNAs exist in cell types other than oocytes.

The Drosophila hairpin RNA pathway generates endogenous short interfering RNAs

In contrast to microRNAs and Piwi-associated RNAs, short interfering RNAs (siRNAs) are seemingly dispensable for host-directed gene regulation in Drosophila. This notion is based on the fact that mutants lacking the core siRNA-generating enzyme Dicer-2 or the predominant siRNA effector Argonaute 2 are viable, fertile and of relatively normal morphology. Moreover, endogenous Drosophila siRNAs have not yet been identified. Here we report that siRNAs derived from long hairpin RNA genes (hpRNAs) programme Slicer complexes that can repress endogenous target transcripts. The Drosophila hpRNA pathway is a hybrid mechanism that combines canonical RNA interference factors (Dicer-2, Hen1 (known as CG12367) and Argonaute 2) with a canonical microRNA factor (Loquacious) to generate approximately 21-nucleotide siRNAs. These novel regulatory RNAs reveal unexpected complexity in the sorting of small RNAs, and open a window onto the biological usage of endogenous RNA interference in Drosophila.

Mammalian Mirtron Genes

Mirtrons are alternative precursors for microRNA biogenesis that were recently described in invertebrates. These short hairpin introns use splicing to bypass Drosha cleavage, which is otherwise essential for the generation of canonical animal microRNAs. Using computational and experimental strategies, we now establish that mammals have mirtrons as well. We identified 3 mirtrons that are well conserved and expressed in diverse mammals, 16 primate-specific mirtrons, and 46 candidates supported by limited cloning evidence in primates. As with some fly and worm mirtrons, the existence of well-conserved mammalian mirtrons indicates their relatively ancient incorporation into endogenous regulatory pathways. However, as worms, flies, and mammals each have different sets of mirtrons, we hypothesize that different animals may have independently evolved the capacity for this hybrid small RNA pathway. This notion is supported by our observation of several clade-specific features of mammalian and invertebrate mirtrons.

The Mirtron Pathway Generates microRNA-Class Regulatory RNAs in Drosophila

The canonical microRNA (miRNA) pathway converts primary hairpin precursor transcripts into approximately 22 nucleotide regulatory RNAs via consecutive cleavages by two RNase III enzymes, Drosha and Dicer. In this study, we characterize Drosophila small RNAs that derive from short intronic hairpins termed "mirtrons." Their nuclear biogenesis appears to bypass Drosha cleavage, which is essential for miRNA biogenesis. Instead, mirtron hairpins are defined by the action of the splicing machinery and lariat-debranching enzyme, which yield pre-miRNA-like hairpins. The mirtron pathway merges with the canonical miRNA pathway during hairpin export by Exportin-5, and both types of hairpins are subsequently processed by Dicer-1/loqs. This generates small RNAs that can repress perfectly matched and seed-matched targets, and we provide evidence that they function, at least in part, via the RNA-induced silencing complex effector Ago1. These findings reveal that mirtrons are an alternate source of miRNA-type regulatory RNAs.