Precursor microRNA-programmed silencing complex assembly pathways in mammals

Assembly of microRNA ribonucleoproteins (miRNPs) or RNA-induced silencing complexes (RISCs) is essential for the function of miRNAs and initiates from processing of precursor miRNAs (pre-miRNAs) by Dicer or by Ago2. Here, we report an in?vitro miRNP/RISC assembly assay programmed by pre-miRNAs from mammalian cell lysates. Combining in?vivo studies in Dicer Knockout cells reconstituted with wild-type or catalytically inactive Dicer, we find that the miRNA loading complex (miRLC) is the primary machinery linking pre-miRNA processing to?miRNA loading. We show that a miRNA precursor deposit complex (miPDC) plays a crucial role in Dicer-independent miRNA biogenesis and promotes?miRNP assembly of certain Dicer-dependent miRNAs. Furthermore, we find that 5'-uridine, 3'-mid base pairing, and 5'-mid mismatches within pre-miRNAs promote their assembly into miPDC. Our studies provide a comprehensive view of miRNP/RISC assembly pathways in mammals, and our assay provides a versatile platform for further mechanistic dissection of such pathways in mammals.     

A New Short Oligonucleotide-Based Strategy for the Precursor-Specific Regulation of microRNA Processing by Dicer

The precise regulation of microRNA (miRNA) biogenesis seems to be critically important for the proper functioning of all eukaryotic organisms. Even small changes in the levels of specific miRNAs can initiate pathological processes, including carcinogenesis. Accordingly, there is a great need to develop effective methods for the regulation of miRNA biogenesis and activity. In this study, we focused on the final step of miRNA biogenesis; i.e., miRNA processing by Dicer. To test our hypothesis that RNA molecules can function not only as Dicer substrates but also as Dicer regulators, we previously identified by SELEX a pool of RNA oligomers that bind to human Dicer. We found that certain of these RNA oligomers could selectively inhibit the formation of specific miRNAs. Here, we show that these specific inhibitors can simultaneously bind both Dicer and pre-miRNAs. These bifunctional riboregulators interfere with miRNA maturation by affecting pre-miRNA structure and sequestering Dicer. Based on these observations, we designed a set of short oligomers (12 nucleotides long) that were capable of influencing pre-miRNA processing in vitro, both in reactions involving recombinant human Dicer and in cytosolic extracts. We propose that the same strategy may be used to develop effective and selective regulators to control the production of any miRNA. Overall, our findings indicate that the interactions between pre-miRNAs and other RNAs may form very complex regulatory networks that modulate miRNA biogenesis and consequently gene expression.     

Derivation and characterization of Dicer- and microRNA-deficient human cells

We have used genome editing to generate inactivating deletion mutations in all three copies of the dicer (hdcr) gene present in the human cell line 293T. As previously shown in murine ES cells lacking Dicer function, hDcr-deficient 293T cells are severely impaired for the production of mature microRNAs (miRNAs). Nevertheless, RNA-induced silencing complexes (RISCs) present in these hDcr-deficient cells are readily programmed by transfected, synthetic miRNA duplexes to repress mRNAs bearing either fully or partially complementary targets, including targets bearing incomplete seed homology to the introduced miRNA. Using these hDcr-deficient 293T cells, we demonstrate that human pre-miRNA processing can be effectively rescued by ectopic expression of the Drosophila Dicer 1 protein, but only in the presence of the PB isoform of Loquacious (Loqs-PB), the fly homolog of the hDcr cofactor TRBP. In contrast, Drosophila Dicer 2, even in the presence of its cofactors Loqs-PD and R2D2, was unable to support human pre-miRNA processing. Interestingly, although ectopic Drosophila Dicer 1/Loqs-PB or hDcr both rescued pre-miRNA processing effectively in these hDcr-deficient cells, there were significant differences in the ratio of the miRNA isoforms that were produced, especially in the case of miR-30 family members, and we also noted differences in the relative expression level of miRNAs vs. passenger strands for a subset of human miRNAs. These data demonstrate that the mechanisms underlying the accurate processing of pre-miRNAs are largely, but not entirely, conserved between mammalian and insect cells.     

Global profiling of miRNAs and the hairpin precursors: insights into miRNA processing and novel miRNA discovery

MicroRNAs (miRNAs) constitute an important class of small regulatory RNAs that are derived from distinct hairpin precursors (pre-miRNAs). In contrast to mature miRNAs, which have been characterized in numerous genome-wide studies of different organisms, research on global profiling of pre-miRNAs is limited. Here, using massive parallel sequencing, we have performed global characterization of both mouse mature and precursor miRNAs. In total, 87 369 704 and 252 003 sequencing reads derived from 887 mature and 281 precursor miRNAs were obtained, respectively. Our analysis revealed new aspects of miRNA/pre-miRNA processing and modification, including eight Ago2-cleaved pre-miRNAs, eight new instances of miRNA editing and exclusively 5′ tailed mirtrons. Furthermore, based on the sequences of both mature and precursor miRNAs, we developed a miRNA discovery pipeline, miRGrep, which does not rely on the availability of genome reference sequences. In addition to 239 known mouse pre-miRNAs, miRGrep predicted 41 novel ones with high confidence. Similar as known ones, the mature miRNAs derived from most of these novel loci showed both reduced abundance following Dicer knockdown and the binding with Argonaute2. Evaluation on data sets obtained from Caenorhabditis elegans and Caenorhabditis sp.11 demonstrated that miRGrep could be widely used for miRNA discovery in metazoans, especially in those without genome reference sequences.     

SNPs in human miRNA genes affect biogenesis and function

MicroRNAs (miRNAs) are 21–25-nucleotide-long, noncoding RNAs that are involved in translational regulation. Most miRNAs derive from a two-step sequential processing: the generation of pre-miRNA from pri-miRNA by the Drosha/DGCR8 complex in the nucleus, and the generation of mature miRNAs from pre-miRNAs by the Dicer/TRBP complex in the cytoplasm. Sequence variation around the processing sites, and sequence variations in the mature miRNA, especially the seed sequence, may have profound affects on miRNA biogenesis and function. In the context of analyzing the roles of miRNAs in Schizophrenia and Autism, we defined at least 24 human X-linked miRNA variants. Functional assays were developed and performed on these variants. In this study we investigate the affects of single nucleotide polymorphisms (SNPs) on the generation of mature miRNAs and their function, and report that naturally occurring SNPs can impair or enhance miRNA processing as well as alter the sites of processing. Since miRNAs are small functional units, single base changes in both the precursor elements as well as the mature miRNA sequence may drive the evolution of new microRNAs by altering their biological function. Finally, the miRNAs examined in this study are X-linked, suggesting that the mutant alleles could be determinants in the etiology of diseases.     

The role of Dicer protein partners in the processing of microRNA precursors

One of the cellular functions of the ribonuclease Dicer is to process microRNA precursors (pre-miRNAs) into mature microRNAs (miRNAs). Human Dicer performs this function in cooperation with its protein partners, AGO2, PACT and TRBP. The exact role of these accessory proteins in Dicer activity is still poorly understood. In this study, we used the northern blotting technique to investigate pre-miRNA cleavage efficiency and specificity after depletion of AGO2, PACT and TRBP by RNAi. The results showed that the inhibition of either Dicer protein partner substantially affected not only miRNA levels but also pre-miRNA levels, and it had a rather minor effect on the specificity of Dicer cleavage. The analysis of the Dicer cleavage products generated in vitro revealed the presence of a cleavage intermediate when pre-miRNA was processed by recombinant Dicer alone. This intermediate was not observed during pre-miRNA cleavage by endogenous Dicer. We demonstrate that AGO2, PACT and TRBP were required for the efficient functioning of Dicer in cells, and we suggest that one of the roles of these proteins is to assure better synchronization of cleavages triggered by two RNase III domains of Dicer.     

Nucleotide sequence of miRNA precursor contributes to cleavage site selection by Dicer

The ribonuclease Dicer excises mature miRNAs from a diverse group of precursors (pre-miRNAs), most of which contain various secondary structure motifs in their hairpin stem. In this study, we analyzed Dicer cleavage in hairpin substrates deprived of such motifs. We searched for the factors other than the secondary structure, which may influence the length diversity and heterogeneity of miRNAs. We found that the nucleotide sequence at the Dicer cleavage site influences both of these miRNA characteristics. With regard to cleavage mechanism, we demonstrate that the Dicer RNase IIIA domain that cleaves within the 3′ arm of the pre-miRNA is more sensitive to the nucleotide sequence of its substrate than is the RNase IIIB domain. The RNase IIIA domain avoids releasing miRNAs with G nucleotide and prefers to generate miRNAs with a U nucleotide at the 5′ end. We also propose that the sequence restrictions at the Dicer cleavage site might be the factor that contributes to the generation of miRNA duplexes with 3′ overhangs of atypical lengths. This finding implies that the two RNase III domains forming the single processing center of Dicer may exhibit some degree of flexibility, which allows for the formation of these non-standard 3′ overhangs.     

Intertwined pathways for Argonaute-mediated microRNA biogenesis in Drosophila

Although Dicer is essential for general microRNA (miRNA) biogenesis, vertebrate mir-451 is Dicer independent. Instead, its short pre-miRNA hairpin is ‘sliced’ by Ago2, then 3′-resected into mature miRNAs. Here, we show that Drosophila cells and animals generate functional small RNAs from mir-451-type precursors. However, their bulk maturation arrests as Ago-cleaved pre-miRNAs, which mostly associate with the RNAi effector AGO2. Routing of pre-mir-451 hairpins to the miRNA effector AGO1 was inhibited by Dicer-1 and its partner Loqs. Loss of these miRNA factors promoted association of pre-mir-451 with AGO1, which sliced them and permitted maturation into ∼23–26 nt products. The difference was due to the 3′ modification of single-stranded species in AGO2 by Hen1 methyltransferase, whose depletion permitted 3′ trimming of Ago-cleaved pre-miRNAs in AGO2. Surprisingly, Nibbler, a 3′–5′ exoribonuclease that trims ‘long’ mature miRNAs in AGO1, antagonized miR-451 processing. We used an in vitro reconstitution assay to identify a soluble, EDTA-sensitive activity that resects sliced pre-miRNAs in AGO1 complexes. Finally, we use deep sequencing to show that depletion of dicer-1 increases the diversity of small RNAs in AGO1, including some candidate mir-451-like loci. Altogether, we document unexpected aspects of miRNA biogenesis and Ago sorting, and provide insights into maturation of Argonaute-cleaved miRNA substrates.     

A universal small molecule,inorganic phosphate,restricts the substrate specificity of Dicer-2 in small RNA biogenesis

The enzyme Dicer is central to the production of small silencing RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs). Like other insects, Drosophila melanogaster uses different Dicers to make siRNAs and miRNAs: Dicer-1 produces miRNAs from pre-miRNAs, whereas Dicer-2 generates siRNAs from long double-stranded RNA (dsRNA). How do the 2 Dicers achieve their substrate specificity? Here, we review recent findings that inorganic phosphate restricts the substrate specificity of Dicer-2 to long dsRNA. Inorganic phosphate inhibits Dicer-2 from binding and cleaving pre-miRNAs, without affecting the processing of long dsRNA. Crystal structures of a fragment of human Dicer in complex with an RNA duplex identify a phosphate-binding pocket that recognizes both the 5′-monophosphate of a substrate RNA and inorganic phosphate. We propose that inorganic phosphate occupies the phosphate-binding pocket in the fly Dicer-2, blocking binding of pre-miRNA and restricting pre-miRNA processing to Dicer-1. Thus, a small molecule can alter the substrate specificity of a nucleic acid-processing enzyme.     

MCPIP1 ribonuclease antagonizes dicer and terminates microRNA biogenesis through precursor microRNA degradation

MicroRNAs (miRNAs) are versatile regulators of gene expression and undergo complex maturation processes. However, the mechanism(s) stabilizing or reducing these small RNAs remains poorly understood. Here we identify mammalian immune regulator MCPIP1 (Zc3h12a) ribonuclease as a broad suppressor of miRNA activity and biogenesis, which counteracts Dicer, a central ribonuclease in miRNA processing. MCPIP1 suppresses miRNA biosynthesis via cleavage of the terminal loops of precursor miRNAs (pre-miRNAs). MCPIP1 also carries a vertebrate-specific oligomerization domain important for pre-miRNA recognition, indicating its recent evolution. Furthermore, we observed potential antagonism between MCPIP1 and Dicer function in human cancer and found a regulatory role of MCPIP1 in the signaling axis comprising miR-155 and its target c-Maf. These results collectively suggest that the balance between processing and destroying ribonucleases modulates miRNA biogenesis and potentially affects pathological miRNA dysregulation. The presence of this abortive processing machinery and diversity of MCPIP1-related genes may imply a dynamic evolutional transition of the RNA silencing system.     

Discovery of novel microRNAs in rat kidney using next generation sequencing and microarray validation

MicroRNAs (miRNAs) are small non-coding RNAs that regulate a variety of biological processes. The latest version of the miRBase database (Release 18) includes 1,157 mouse and 680 rat mature miRNAs. Only one new rat mature miRNA was added to the rat miRNA database from version 16 to version 18 of miRBase, suggesting that many rat miRNAs remain to be discovered. Given the importance of rat as a model organism, discovery of the completed set of rat miRNAs is necessary for understanding rat miRNA regulation. In this study, next generation sequencing (NGS), microarray analysis and bioinformatics technologies were applied to discover novel miRNAs in rat kidneys. MiRanalyzer was utilized to analyze the sequences of the small RNAs generated from NGS analysis of rat kidney samples. Hundreds of novel miRNA candidates were examined according to the mappings of their reads to the rat genome, presence of sequences that can form a miRNA hairpin structure around the mapped locations, Dicer cleavage patterns, and the levels of their expression determined by both NGS and microarray analyses. Nine novel rat hairpin precursor miRNAs (pre-miRNA) were discovered with high confidence. Five of the novel pre-miRNAs are also reported in other species while four of them are rat specific. In summary, 9 novel pre-miRNAs (14 novel mature miRNAs) were identified via combination of NGS, microarray and bioinformatics high-throughput technologies.     

Monocyte chemoattractant protein-induced protein 1 directly degrades viral miRNAs with a specific motif and inhibits KSHV infection

Kaposi''s sarcoma-associated herpesvirus (KSHV) expresses miRNAs during latency. However, regulation of viral miRNAs remains largely unknown. Our prior studies demonstrated that MCPIP1 regulates KSHV miRNA biogenesis by degrading most KSHV pre-miRNAs through its RNase activity. Some viral pre-miRNAs are partially resistant to degradation by MCPIP1. Here, we further characterized MCPIP1 substrate specificity and its antiviral potential against KSHV infection. In vitro cleavage assays and binding assays showed that MCPIP1 cleavage efficiency is related to binding affinity. Motif-based sequence analysis identified that KSHV pre-miRNAs that are well degraded by MCPIP1 have a 5-base motif (M5 base motif) within their terminal loops and this motif region consists of multiple pyrimidine-purine-pyrimidine (YRY) motifs. We further demonstrated that mutation of this M5 base motif within terminal loop of pre-miRNAs inhibited MCPIP1-mediated RNA degradation. We also revealed that MCPIP1 has an antiviral effect against KSHV infection. MCPIP1 can reduce the expression of Dicer, which in turn restricts KSHV infection. Conclusively, our findings demonstrated that MCPIP1 inhibited KSHV infection and suppressed viral miRNA biogenesis by directly degrading KSHV pre-miRNAs and altering the expression of miRNA biogenesis factors.