Noncanonical processing by animal Microprocessor.

Microprocessor (MP), DROSHA-DGCR8, processes primary miRNA transcripts (pri-miRNAs) to initiate miRNA biogenesis. The canonical cleavage mechanism of MP has been extensively investigated and comprehensively validated for two decades. However, this canonical mechanism cannot account for the processing of certain pri-miRNAs in animals. In this study, by conducting high-throughput pri-miRNA cleavage assays for approximately 260,000 pri-miRNA sequences, we discovered and comprehensively characterized a noncanonical cleavage mechanism of MP. This noncanonical mechanism does not need several RNA and protein elements essential for the canonical mechanism; instead, it utilizes previously unrecognized DROSHA dsRNA recognition sites (DRESs). Interestingly, the noncanonical mechanism is conserved across animals and plays a particularly significant role in C. elegans. Our established noncanonical mechanism elucidates MP cleavage in numerous RNA substrates unaccounted for by the canonical mechanism in animals. This study suggests a broader substrate repertoire of animal MPs and an expanded regulatory landscape for miRNA biogenesis.

A quantitative map of human primary microRNA processing sites.

Maturation of canonical microRNA (miRNA) is initiated by DROSHA that cleaves the primary transcript (pri-miRNA). More than 1,800 miRNA loci are annotated in humans, but it remains largely unknown whether and at which sites pri-miRNAs are cleaved by DROSHA. Here, we performed in vitro processing on a full set of human pri-miRNAs (miRBase version 21) followed by sequencing. This comprehensive profiling enabled us to classify miRNAs on the basis of DROSHA dependence and map their cleavage sites with respective processing efficiency measures. Only 758 pri-miRNAs are confidently processed by DROSHA, while the majority may be non-canonical or false entries. Analyses of the DROSHA-dependent pri-miRNAs show key cis-elements for processing. We observe widespread alternative processing and unproductive cleavage events such as "nick" or "inverse" processing. SRSF3 is a broad-acting auxiliary factor modulating alternative processing and suppressing unproductive processing. The profiling data and methods developed in this study will allow systematic analyses of miRNA regulation.

MicroRNA Clustering Assists Processing of Suboptimal MicroRNA Hairpins through the Action of the ERH Protein.

Microprocessor initiates the processing of microRNAs (miRNAs) from the hairpin regions of primary transcripts (pri-miRNAs). Pri-miRNAs often contain multiple miRNA hairpins, and this clustered arrangement can assist in the processing of otherwise defective hairpins. We find that miR-451, which derives from a hairpin with a suboptimal terminal loop and a suboptimal stem length, accumulates to 40-fold higher levels when clustered with a helper hairpin. This phenomenon tolerates changes in hairpin order, linker lengths, and the identities of the helper hairpin, the recipient hairpin, the linker-sequence, and the RNA polymerase that transcribes the hairpins. It can act reciprocally and need not occur co-transcriptionally. It requires Microprocessor recognition of the helper hairpin and linkage of the two hairpins, yet predominantly manifests after helper-hairpin processing. It also requires enhancer of rudimentary homolog (ERH), which copurifies with Microprocessor and can dimerize and interact with other proteins that can dimerize, suggesting a model in which one Microprocessor recruits another Microprocessor.

SAFB2 Enables the Processing of Suboptimal Stem-Loop Structures in Clustered Primary miRNA Transcripts.

Many microRNAs (miRNAs) are generated from primary transcripts containing multiple clustered stem-loop structures that are thought to be recognized and cleaved by the Microprocessor complex as independent units. Here, we uncover an unexpected mode of processing of the bicistronic miR-15a-16-1 cluster. We find that the primary miR-15a stem-loop is not processed on its own but that the presence of the neighboring primary miR-16-1 stem-loop on the same transcript can compensate for this deficiency in cis. Using a CRISPR/Cas9 screen, we identify SAFB2 (scaffold attachment factor B2) as an essential co-factor in this miR-16-1-assisted pri-miR-15 cleavage and describe SAFB2 as an accessory protein of the Microprocessor. Notably, SAFB2-mediated cleavage expands to other clustered pri-miRNAs, indicating a general mechanism. Together, our study reveals an unrecognized function of SAFB2 in miRNA processing and suggests a scenario in which SAFB2 enables the binding and processing of suboptimal Microprocessor substrates in clustered primary miRNA transcripts.

SRSF3 shapes the structure of miR-17-92 cluster RNA and promotes selective processing of miR-17 and miR-20a.

MicroRNA (miRNA) biogenesis is tightly regulated to maintain distinct miRNA expression patterns. Almost half of mammalian miRNAs are generated from miRNA clusters, but this process is not well understood. We show here that Serine-arginine rich splicing factor 3 (SRSF3) controls the processing of miR-17-92 cluster miRNAs in pluripotent and cancer cells. SRSF3 binding to multiple CNNC motifs downstream of Drosha cleavage sites within miR-17-92 is required for the efficient processing of the cluster. SRSF3 depletion specifically compromises the processing of two paralog miRNAs, miR-17 and miR-20a. In addition to SRSF3 binding to the CNNC sites, the SRSF3 RS-domain is essential for miR-17-92 processing. SHAPE-MaP probing demonstrates that SRSF3 binding disrupts local and distant base pairing, resulting in global changes in miR-17-92 RNA structure. Our data suggest a model where SRSF3 binding, and potentially its RS-domain interactions, may facilitate an RNA structure that promotes miR-17-92 processing. SRSF3-mediated increase in miR-17/20a levels inhibits the cell cycle inhibitor p21, promoting self-renewal in normal and cancer cells. The SRSF3-miR-17-92-p21 pathway operates in colorectal cancer, linking SRSF3-mediated pri-miRNA processing and cancer pathogenesis.

N4-acetylcytidine modifies primary microRNAs for processing in cancer cells.

N4 acetylcytidine (ac4C) modification mainly occurs on tRNA, rRNA, and mRNA, playing an important role in the expression of genetic information. However, it is still unclear whether microRNAs have undergone ac4C modification and their potential physiological and pathological functions. In this study, we identified that NAT10/THUMPD1 acetylates primary microRNAs (pri-miRNAs) with ac4C modification. Knockdown of NAT10 suppresses and augments the expression levels of mature miRNAs and pri-miRNAs, respectively. Molecular mechanism studies found that pri-miRNA ac4C promotes the processing of pri-miRNA into precursor miRNA (pre-miRNA) by enhancing the interaction of pri-miRNA and DGCR8, thereby increasing the biogenesis of mature miRNA. Knockdown of NAT10 attenuates the oncogenic characters of lung cancer cells by regulating miRNA production in cancers. Moreover, NAT10 is highly expressed in various clinical cancers and negatively correlated with poor prognosis. Thus, our results reveal that NAT10 plays a crucial role in cancer initiation and progression by modulating pri-miRNA ac4C to affect miRNA production, which would provide an attractive therapeutic strategy for cancers.

Nuclear receptor RXRα binds the precursor of miR-103 to inhibit its maturation.

BACKGROUND: The maturation of microRNAs (miRNAs) successively undergoes Drosha, Dicer, and Argonaute -mediated processing, however, the intricate regulations of the individual miRNA maturation are largely unknown. Retinoid x receptor alpha (RXRα) belongs to nuclear receptors that regulate gene transcription by binding to DNA elements, however, whether RXRα binds to miRNAs to exert physiological functions is not known. RESULTS: In this work, we found that RXRα directly binds to the precursor of miR-103 (pre-miR-103a-2) via its DNA-binding domain with a preferred binding sequence of AGGUCA. The binding of RXRα inhibits the processing of miR-103 maturation from pre-miR-103a-2. Mechanistically, RXRα prevents the nuclear export of pre-miR-103a-2 for further processing by inhibiting the association of exportin-5 with pre-miR-103a-2. Pathophysiologically, the negative effect of RXRα on miR-103 maturation correlates to the positive effects of RXRα on the expression of Dicer, a target of miR-103, and on the inhibition of breast cancer. CONCLUSIONS: Our findings unravel an unexpected role of transcription factor RXRα in specific miRNA maturation at post-transcriptional level through pre-miRNA binding, and present a mechanistic insight regarding RXRα role in breast cancer progression.

miRNA- and Cell Line-Specific Constraints on Precursor miRNA Processing of Stably Transfected Pancreatic Cancer and Other Mammalian Cells.

MicroRNAs (miRNAs) regulate approximately one-third of all human genes. The dysregulation of miRNAs has been implicated in the development of numerous human diseases, including cancers. In our investigation focusing on altering specific miRNA expression in human pancreatic cancer cells, we encountered an interesting finding. While two expression vector designs effectively enhanced miR-708 levels, they were unable to elevate mature forms of miR-29b, -1290, -2467, and -6831 in pancreatic cancer cell lines. This finding was also observed in a panel of other non-pancreatic cancer cell lines, suggesting that miRNA processing efficiency was cell line specific. Using a step-by-step approach in each step of miRNA processing, we ruled out alternative strand selection by the RISC complex and transcriptional interference at the primary miRNA (pri-miRNA) level. DROSHA processing and pri-miRNA export from the nucleus also appeared to be occurring normally. We observed precursor (pre-miRNA) accumulation only in cell lines where mature miRNA expression was not achieved, suggesting that the block was occurring at the pre-miRNA stage. To further confirm this, synthetic pre-miRNA mimics that bypass DICER processing were processed into mature miRNAs in all cases. This study has demonstrated the distinct behaviours of different miRNAs with the same vector in the same cell line, the same miRNA between the two vector designs, and with the same miRNA across different cell lines. We identified a stable vector pre-miRNA processing block. Our findings on the structural and sequence differences between successful and non-successful vector designs could help to inform future chimeric miRNA design strategies and act as a guide to other researchers on the intricate processing dynamics that can impact vector efficiency. Our research confirms the potential of miRNA mimics to surmount some of these complexities.

Keeping up with the miRNAs: current paradigms of the biogenesis pathway.

For many years we have studied the processes involved in producing miRNAs in plants and the numerous differences from their metazoan counterpart. A well-defined catalytic process, mostly carried out by the RNase III enzyme DICER-LIKE1 (DCL1), it was identified early after the discovery of RNAi and was followed by the isolation of a plethora of miRNA biogenesis cofactors. The production of miRNAs, which later are loaded in ARGONAUTE (AGO) proteins to perform their RNA silencing functions both within the cell and non-cell autonomously, appears to be a highly regulated and dynamic process. Many regulatory events during miRNA biogenesis require the action of specific proteins. However, in recent years, many post-transcriptional modifications, structural features, and coupling with other cellular processing emerged as critical elements controlling the production of miRNA and, thus, a plant's physiology. This review discusses new evidence that has changed the way we understand how miRNAs are produced in plants. We also provide an updated view of the miRNA biogenesis pathways, focusing on the gaps in our knowledge and the most compelling questions that remain open.

DGCR8-dependent efficient pri-miRNA processing of human pri-miR-9-2.

Microprocessor complex, including DiGeorge syndrome critical region gene 8 (DGCR8) and DROSHA, recognizes and cleaves primary transcripts of microRNAs (pri-miRNAs) in the maturation of canonical miRNAs. The study of DGCR8 haploinsufficiency reveals that the efficiency of this activity varies for different miRNA species. It is thought that this variation might be associated with the risk of schizophrenia with 22q11 deletion syndrome caused by disruption of the DGCR8 gene. However, the underlying mechanism for varying action of DGCR8 with each miRNA remains largely unknown. Here, we used in vivo monitoring to measure the efficiency of DGCR8-dependent microprocessor activity in cultured cells. We confirmed that this system recapitulates the microprocessor activity of endogenous pri-miRNA with expression of a ratiometric fluorescence reporter. Using this system, we detected mir-9-2 as one of the most efficient targets. We also identified a novel DGCR8-responsive RNA element, which is highly conserved among mammalian species and could be regulated at the epi-transcriptome (RNA modification) level. This unique feature between DGCR8 and pri-miR-9-2 processing may suggest a link to the risk of schizophrenia.

Challenging Cellular Homeostasis: Spatial and Temporal Regulation of miRNAs.

Mature microRNAs (miRNAs) are single-stranded non-coding RNA (ncRNA) molecules that act in post-transcriptional regulation in animals and plants. A mature miRNA is the end product of consecutive, highly regulated processing steps of the primary miRNA transcript. Following base-paring of the mature miRNA with its mRNA target, translation is inhibited, and the targeted mRNA is degraded. There are hundreds of miRNAs in each cell that work together to regulate cellular key processes, including development, differentiation, cell cycle, apoptosis, inflammation, viral infection, and more. In this review, we present an overlooked layer of cellular regulation that addresses cell dynamics affecting miRNA accessibility. We discuss the regulation of miRNA local storage and translocation among cell compartments. The local amounts of the miRNAs and their targets dictate their actual availability, which determines the ability to fine-tune cell responses to abrupt or chronic changes. We emphasize that changes in miRNA storage and compactization occur under induced stress and changing conditions. Furthermore, we demonstrate shared principles on cell physiology, governed by miRNA under oxidative stress, tumorigenesis, viral infection, or synaptic plasticity. The evidence presented in this review article highlights the importance of spatial and temporal miRNA regulation for cell physiology. We argue that limiting the research to mature miRNAs within the cytosol undermines our understanding of the efficacy of miRNAs to regulate cell fate under stress conditions.

Sex-specific, reciprocal regulation of ERα and miR-22 controls muscle lipid metabolism in male mice.

Control of energy homeostasis and metabolism is achieved by integrating numerous pathways, and miRNAs are involved in this process by regulating expression of multiple target genes. However, relatively little is known about the posttranscriptional processing of miRNAs and a potential role for the precursors they derive from. Here, we demonstrate that mature miRNA-22 is more abundant in muscle from male mice relative to females and that this enables sex-specific regulation of muscular lipid metabolism and body weight by repressing estrogen receptor alpha (ERα) expression. We found that the ERα adjusts its own activity by preventing processing of miR-22 via direct binding to a conserved ERα-binding element within the primary miR-22 precursor. Mutation of the ERα binding site within the pri-miR-22 in vivo eliminates sex-specific differences in miR-22 expression. We reason that the resulting tissue selective negative feedback regulation is essential to establish sex-specific differences in muscle metabolism and body weight development.

Recent advances in the regulation of plant miRNA biogenesis.

MicroRNAs (miRNAs) are essential non-coding riboregulators of gene expression in plants and animals. In plants, miRNAs guide their effector protein named ARGONAUTE (AGO) to find target RNAs for gene silencing through target RNA cleavage or translational inhibition. miRNAs are derived from primary miRNA transcripts (pri-miRNAs), most of which are transcribed by the DNA-dependent RNA polymerase II. In plants, an RNase III enzyme DICER-LIKE1-containing complex processes pri-miRNAs in the nucleus into miRNAs. To ensure proper function of miRNAs, plants use multiple mechanisms to control miRNA accumulation. On one hand, pri-miRNA levels are controlled through transcription and stability. On the other hand, the activities of the DCL1 complex are regulated by many protein factors at transcriptional, post-transcriptional and post-translational levels. Notably, recent studies reveal that pri-miRNA structure/sequence features and modifications also play important roles in miRNA biogenesis. In this review, we summarize recent progresses on the mechanisms regulating miRNA biogenesis.

Splicing factors as regulators of miRNA biogenesis - links to human disease.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post-transcriptionally by fine-tuning mRNA levels and translation during development and in adult tissues. miRNAs are transcribed as parts of longer precursors that undergo multiple processing steps before the mature miRNAs reach their target mRNAs in the cytoplasm. In addition to Drosha/DGCR8 and Dicer that are the essential components of the miRNA processing pathway, a range of other RNA binding proteins have recently been implicated in miRNA biogenesis. Among these, several well-known splicing factors have emerged as regulators of distinct miRNAs. In this review, we examine the mechanisms by which splicing factors regulate miRNA biogenesis. As both splicing factors and miRNAs play central roles in human disease biology we discuss implications of the links between splicing factors and miRNAs in human disease.

Investigation into the miRNA/5' isomiRNAs function and drought-mediated miRNA processing in rice.

MicroRNAs lie at the core of biological regulatory networks in plants. The recent discovery of isomiRs that are length variants of the annotated mature miRNAs has further unveiled the complexity of miRNome. Delineation of their functional relevance is critical to understand the complete functional spectrum of the miRNome. To apprehend the role of 5' isomiRs in rice, we performed a comprehensive analysis of the annotated miRNA pool using 8 deep-sequencing datasets from flag leaf and spikelet tissues from two cultivars of rice viz. N22 and PB1 grown under control and drought conditions. The products of the 5' start site variability termed as "5' isomiRs" were found to be widespread in all the datasets. It was possible to identify several 5' isomiRs that were highly distinct and abundant and supported by more than 90% of the tags that map in the region. Majority of miRNA/5' isomiR pair share similar tissue and drought-mediated expression dynamics. Analysis of the degradome data identified targets for several of these 5' isomiRs, thereby confirming their biological activity. Since the isomiRs are length variants at the 5' end, the target sites were found to be accordingly shifted as compared to the target site of the annotated miRNA. Further we also observed that drought affects the processing accuracy of several miRNAs across all tissues of both the cultivars leading to differential accumulation of 5' isomiR/miRNA pair.

Tankyrase promotes primary precursor miRNA processing to precursor miRNA.

Tankyrases (TNKS and TNKS2) are members of poly(ADP-ribose) polymerase (PARP) family proteins. Tankyrase has multiple ankyrin repeat cluster (ARC) domains, which recognize the tankyrase-binding motifs in proteins including the telomeric protein, TRF1 and Wnt signal regulators, AXINs. However, the functional significance of tankyrase interaction with many other putative binding proteins remains unknown. Here, we found that several proteins involved in microRNA (miRNA) processing have putative tankyrase-binding motifs and their functions are regulated by tankyrase. First, chemical inhibition of tankyrase PARP activity downregulated the expression levels of precursor miRNAs (pre-miRNAs) but not primary precursor miRNAs (pri-miRNAs). A subsequent reporter assay revealed that tankyrase inhibitors or PARP-dead mutant tankyrase overexpression repress pri-miRNA processing to pre-miRNA. Conversely, a PARP-1/2 inhibitor, olaparib, did not affect pri-miRNA processing. Tankyrase ARCs bound to DGCR8 and DROSHA, which are essential components for pri-miRNA processing and have putative tankyrase-binding motifs. These observations indicate that tankyrase binds to Microprocessor, DGCR8 and DROSHA complex and modulates pri-miRNA processing to pre-miRNA.

Molecular subgrouping of primary pineal parenchymal tumors reveals distinct subtypes correlated with clinical parameters and genetic alterations.

Tumors of the pineal region comprise several different entities with distinct clinical and histopathological features. Whereas some entities predominantly affect adults, pineoblastoma (PB) constitutes a highly aggressive malignancy of childhood with a poor outcome. PBs mainly arise sporadically, but may also occur in the context of cancer predisposition syndromes including DICER1 and RB1 germline mutation. With this study, we investigate clinico-pathological subgroups of pineal tumors and further characterize their biological features. We performed genome-wide DNA methylation analysis in 195 tumors of the pineal region and 20 normal pineal gland controls. Copy-number profiles were obtained from DNA methylation data; gene panel sequencing was added for 93 tumors and analysis was further complemented by miRNA sequencing for 22 tumor samples. Unsupervised clustering based on DNA methylation profiling separated known subgroups, like pineocytoma, pineal parenchymal tumor of intermediate differentiation, papillary tumor of the pineal region and PB, and further distinct subtypes within these groups, including three subtypes within the core PB subgroup. The novel molecular subgroup Pin-RB includes cases of trilateral retinoblastoma as well as sporadic pineal tumors with RB1 alterations, and displays similarities with retinoblastoma. Distinct clinical associations discriminate the second novel molecular subgroup PB-MYC from other PB cases. Alterations within the miRNA processing pathway (affecting DROSHA, DGCR8 or DICER1) are found in about two thirds of cases in the three core PB subtypes. Methylation profiling revealed biologically distinct groups of pineal tumors with specific clinical and molecular features. Our findings provide a foundation for further clinical as well as molecular and functional characterization of PB and other pineal tumors, including the role of miRNA processing defects in oncogenesis.

The dynamic recruitment of TRBP to neuronal membranes mediates dendritogenesis during development.

MicroRNAs are important regulators of local protein synthesis during neuronal development. We investigated the dynamic regulation of microRNA production and found that the majority of the microRNA-generating complex, consisting of Dicer, TRBP, and PACT, specifically associates with intracellular membranes in developing neurons. Stimulation with brain-derived neurotrophic factor (BDNF), which promotes dendritogenesis, caused the redistribution of TRBP from the endoplasmic reticulum into the cytoplasm, and its dissociation from Dicer, in a Ca2+-dependent manner. As a result, the processing of a subset of neuronal precursor microRNAs, among them the dendritically localized pre-miR16, was impaired. Decreased production of miR-16-5p, which targeted the BDNF mRNA itself, was rescued by expression of a membrane-targeted TRBP Moreover, miR-16-5p or membrane-targeted TRBP expression blocked BDNF-induced dendritogenesis, demonstrating the importance of neuronal TRBP dynamics for activity-dependent neuronal development. We propose that neurons employ specialized mechanisms to modulate local gene expression in dendrites, via the dynamic regulation of microRNA biogenesis factors at intracellular membranes of the endoplasmic reticulum, which in turn is crucial for neuronal dendrite complexity and therefore neuronal circuit formation and function.

KETCH1 imports HYL1 to nucleus for miRNA biogenesis in Arabidopsis.

MicroRNA (miRNA) is processed from primary transcripts with hairpin structures (pri-miRNAs) by microprocessors in the nucleus. How cytoplasmic-borne microprocessor components are transported into the nucleus to fulfill their functions remains poorly understood. Here, we report KETCH1 (karyopherin enabling the transport of the cytoplasmic HYL1) as a partner of hyponastic leaves 1 (HYL1) protein, a core component of microprocessor in Arabidopsis and functional counterpart of DGCR8/Pasha in animals. Null mutation of ketch1 is embryonic-lethal, whereas knockdown mutation of ketch1 caused morphological defects, reminiscent of mutants in the miRNA pathway. ketch1 knockdown mutation also substantially reduced miRNA accumulation, but did not alter nuclear-cytoplasmic shuttling of miRNAs. Rather, the mutation significantly reduced nuclear portion of HYL1 protein and correspondingly compromised the pri-miRNA processing in the nucleus. We propose that KETCH1 transports HYL1 from the cytoplasm to the nucleus to constitute functional microprocessor in Arabidopsis This study provides insight into the largely unknown nuclear-cytoplasmic trafficking process of miRNA biogenesis components through eukaryotes.

Conserved and novel roles of miR164-CUC2 regulatory module in specifying leaf and floral organ morphology in strawberry.

MicroRNAs (miRNAs) are a kind of short noncoding RNA (20-24 nt), playing versatile roles in plant growth and development. Strawberry generates leaves and flowers with unique features. However, few miRNAs have been functionally characterised in strawberry, especially for their developmental regulation. Here, we identified one ethyl methanesulfonate (EMS) mutant, deeply serrated (des), in the woodland strawberry Fragaria vesca that has wrinkled leaves with deeper serrations, serrated petals and deformed carpels. The causative mutation occurs in the 19th nucleotide of the FvemiR164a mature sequence. Overexpressing FveMIR164A rescued the phenotypes of des/fvemir164a except the petal serrations. Furthermore, we identified two allelic mutants of FveCUC2a, one target of FvemiR164a, which developed leaves with smooth margins and fused leaflets. Phenotypes of the double mutant fvemir164a fvecuc2a indicated that the two genes act linearly in leaf and carpel development, but synergistically in the development of other floral organs and inflorescence architecture. This work demonstrates the conserved and novel roles of the miR164-CUC2 module in leaf and flower development in different plant species, and reveals that the 19th nucleotide of FvemiR164a is important for its processing.