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.

Structural Basis for pri-miRNA Recognition by Drosha.

A commencing and critical step in miRNA biogenesis involves processing of pri-miRNAs in the nucleus by Microprocessor. An important, but not completely understood, question is how Drosha, the catalytic subunit of Microprocessor, binds pri-miRNAs and correctly specifies cleavage sites. Here we report the cryoelectron microscopy structures of the Drosha-DGCR8 complex with and without a pri-miRNA. The RNA-bound structure provides direct visualization of the tertiary structure of pri-miRNA and shows that a helix hairpin in the extended PAZ domain and the mobile basic (MB) helix in the RNase IIIa domain of Drosha coordinate to recognize the single-stranded to double-stranded junction of RNA, whereas the dsRNA binding domain makes extensive contacts with the RNA stem. Furthermore, the RNA-free structure reveals an autoinhibitory conformation of the PAZ helix hairpin. These findings provide mechanistic insights into pri-miRNA cleavage site selection and conformational dynamics governing pri-miRNA recognition by the catalytic component of Microprocessor.

Cryo-EM Structures of Human Drosha and DGCR8 in Complex with Primary MicroRNA.

Metazoan microRNAs require specific maturation steps initiated by Microprocessor, comprising Drosha and DGCR8. Lack of structural information for the assembled complex has hindered an understanding of how Microprocessor recognizes primary microRNA transcripts (pri-miRNAs). Here we present a cryoelectron microscopy structure of human Microprocessor with a pri-miRNA docked in the active site, poised for cleavage. The basal junction is recognized by a four-way intramolecular junction in Drosha, triggered by the Belt and Wedge regions that clamp over the ssRNA. The belt is important for efficiency and accuracy of pri-miRNA processing. Two dsRBDs form a molecular ruler to measure the stem length between the two dsRNA-ssRNA junctions. The specific organization of the dsRBDs near the apical junction is independent of Drosha core domains, as observed in a second structure in the partially docked state. Collectively, we derive a molecular model to explain how Microprocessor recognizes a pri-miRNA and accurately identifies the cleavage site.

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.

Functional Atlas of Primary miRNA Maturation by the Microprocessor.

Primary microRNAs (miRNAs) are the precursors of miRNAs that modulate the expression of most mRNAs in humans. They fold up into a hairpin structure that is cleaved at its base by an enzyme complex known as the Microprocessor (Drosha/DGCR8). While many of the molecular details are known, a complete understanding of what features distinguish primary miRNA from hairpin structures in other transcripts is still lacking. We develop a massively parallel functional assay termed Dro-seq (Drosha sequencing) that enables testing of hundreds of known primary miRNA substrates and thousands of single-nucleotide variants. We find an additional feature of primary miRNAs, called Shannon entropy, describing the structural ensemble important for processing. In a deep mutagenesis experiment, we observe particular apical loop U bases, likely recognized by DGCR8, are important for efficient processing. These findings build on existing knowledge about primary miRNA maturation by the Microprocessor and further explore the substrate RNA sequence-structure relationship.

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.

Genome-wide Mapping of DROSHA Cleavage Sites on Primary MicroRNAs and Noncanonical Substrates.

MicroRNA (miRNA) maturation is initiated by DROSHA, a double-stranded RNA (dsRNA)-specific RNase III enzyme. By cleaving primary miRNAs (pri-miRNAs) at specific positions, DROSHA serves as a main determinant of miRNA sequences and a highly selective gatekeeper for the canonical miRNA pathway. However, the sites of DROSHA-mediated processing have not been annotated, and it remains unclear to what extent DROSHA functions outside the miRNA pathway. Here, we establish a protocol termed "formaldehyde crosslinking, immunoprecipitation, and sequencing (fCLIP-seq)," which allows identification of DROSHA cleavage sites at single-nucleotide resolution. fCLIP identifies numerous processing sites, suggesting widespread end modifications during miRNA maturation. fCLIP also finds many pri-miRNAs that undergo alternative processing, yielding multiple miRNA isoforms. Moreover, we discovered dozens of DROSHA substrates on non-miRNA loci, which may serve as cis-elements for DROSHA-mediated gene regulation. We anticipate that fCLIP-seq could be a general tool for investigating interactions between dsRNA-binding proteins and structured RNAs.

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.

Arabidopsis AAR2, a conserved splicing factor in eukaryotes, acts in microRNA biogenesis.

MicroRNAs (miRNAs) play an essential role in plant growth and development, and as such, their biogenesis is fine-tuned via regulation of the core microprocessor components. Here, we report that Arabidopsis AAR2, a homolog of a U5 snRNP assembly factor in yeast and humans, not only acts in splicing but also promotes miRNA biogenesis. AAR2 interacts with the microprocessor component hyponastic leaves 1 (HYL1) in the cytoplasm, nucleus, and dicing bodies. In aar2 mutants, abundance of nonphosphorylated HYL1, the active form of HYL1, and the number of HYL1-labeled dicing bodies are reduced. Primary miRNA (pri-miRNA) accumulation is compromised despite normal promoter activities of MIR genes in aar2 mutants. RNA decay assays show that the aar2-1 mutation leads to faster degradation of pri-miRNAs in a HYL1-dependent manner, which reveals a previously unknown and negative role of HYL1 in miRNA biogenesis. Taken together, our findings reveal a dual role of AAR2 in miRNA biogenesis and pre-messenger RNA splicing.

Intramolecular ligation method (iLIME) for pre-miRNA quantification and sequencing.

Hairpin-containing pre-miRNAs, produced from pri-miRNAs, are precursors of miRNAs (microRNAs) that play essential roles in gene expression and various human diseases. Current qPCR-based methods used to quantify pre-miRNAs are not effective to discriminate between pre-miRNAs and their parental pri-miRNAs. Here, we developed the intramolecular ligation method (iLIME) to quantify and sequence pre-miRNAs specifically. This method utilizes T4 RNA ligase 1 to convert pre-miRNAs into circularized RNAs, allowing us to design PCR primers to quantify pre-miRNAs, but not their parental pri-miRNAs. In addition, the iLIME also enables us to sequence the ends of pre-miRNAs using next-generation sequencing. Therefore, this method offers a simple and effective way to quantify and sequence pre-miRNAs, so it will be highly beneficial for investigating pre-miRNAs when addressing research questions and medical applications.

MiRNAs differentially expressed in vegetative and reproductive organs of Marchantia polymorpha - insights into their expression pattern, gene structures and function.

MicroRNAs regulate gene expression affecting a variety of plant developmental processes. The evolutionary position of Marchantia polymorpha makes it a significant model to understand miRNA-mediated gene regulatory pathways in plants. Previous studies focused on conserved miRNA-target mRNA modules showed their critical role in Marchantia development. Here, we demonstrate that the differential expression of conserved miRNAs among land plants and their targets in selected organs of Marchantia additionally underlines their role in regulating fundamental developmental processes. The main aim of this study was to characterize selected liverwort-specific miRNAs, as there is a limited knowledge on their biogenesis, accumulation, targets, and function in Marchantia. We demonstrate their differential accumulation in vegetative and generative organs. We reveal that all liverwort-specific miRNAs examined are encoded by independent transcriptional units. MpmiR11737a, MpmiR11887 and MpmiR11796, annotated as being encoded within protein-encoding genes, have their own independent transcription start sites. The analysis of selected liverwort-specific miRNAs and their pri-miRNAs often reveal correlation in their levels, suggesting transcriptional regulation. However, MpmiR11796 shows a reverse correlation to its pri-miRNA level, suggesting post-transcriptional regulation. Moreover, we identify novel targets for selected liverwort-specific miRNAs and demonstrate an inverse correlation between their expression and miRNA accumulation. In the case of one miRNA precursor, we provide evidence that it encodes two functional miRNAs with two independent targets. Overall, our research sheds light on liverwort-specific miRNA gene structure, provides new data on their biogenesis and expression regulation. Furthermore, identifying their targets, we hypothesize the potential role of these miRNAs in early land plant development and functioning.

HP1BP3, a Chromatin Retention Factor for Co-transcriptional MicroRNA Processing.

Recent studies suggest that the microprocessor (Drosha-DGCR8) complex can be recruited to chromatin to catalyze co-transcriptional processing of primary microRNAs (pri-miRNAs) in mammalian cells. However, the molecular mechanism of co-transcriptional miRNA processing is poorly understood. Here we find that HP1BP3, a histone H1-like chromatin protein, specifically associates with the microprocessor and promotes global miRNA biogenesis in human cells. Chromatin immunoprecipitation (ChIP) studies reveal genome-wide co-localization of HP1BP3 and Drosha and HP1BP3-dependent Drosha binding to actively transcribed miRNA loci. Moreover, HP1BP3 specifically binds endogenous pri-miRNAs and facilitates the Drosha/pri-miRNA association in vivo. Knockdown of HP1BP3 compromises pri-miRNA processing by causing premature release of pri-miRNAs from the chromatin. Taken together, these studies suggest that HP1BP3 promotes co-transcriptional miRNA processing via chromatin retention of nascent pri-miRNA transcripts. This work significantly expands the functional repertoire of the H1 family of proteins and suggests the existence of chromatin retention factors for widespread co-transcriptional miRNA processing.

miRNA-independent function of long noncoding pri-miRNA loci.

Among the large, diverse set of mammalian long noncoding RNAs (lncRNAs), long noncoding primary microRNAs (lnc-pri-miRNAs) are those that host miRNAs. Whether lnc-pri-miRNA loci have important biological function independent of their cognate miRNAs is poorly understood. From a genome-scale lncRNA screen, lnc-pri-miRNA loci were enriched for function in cell proliferation, and in glioblastoma (i.e., GBM) cells with DGCR8 or DROSHA knockdown, lnc-pri-miRNA screen hits still regulated cell growth. To molecularly dissect the function of a lnc-pri-miRNA locus, we studied LOC646329 (also known as MIR29HG), which hosts the miR-29a/b1 cluster. In GBM cells, LOC646329 knockdown reduced miR-29a/b1 levels, and these cells exhibited decreased growth. However, genetic deletion of the miR-29a/b1 cluster (LOC646329-miR29Δ) did not decrease cell growth, while knockdown of LOC646329-miR29Δ transcripts reduced cell proliferation. The miR-29a/b1-independent activity of LOC646329 corresponded to enhancer-like activation of a neighboring oncogene (MKLN1), regulating cell propagation. The LOC646329 locus interacts with the MKLN1 promoter, and antisense oligonucleotide knockdown of the lncRNA disrupts these interactions and reduces the enhancer-like activity. More broadly, analysis of genome-wide data from multiple human cell types showed that lnc-pri-miRNA loci are significantly enriched for DNA looping interactions with gene promoters as well as genomic and epigenetic characteristics of transcriptional enhancers. Functional studies of additional lnc-pri-miRNA loci demonstrated cognate miRNA-independent enhancer-like activity. Together, these data demonstrate that lnc-pri-miRNA loci can regulate cell biology via both miRNA-dependent and miRNA-independent mechanisms.

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.

MAC5, an RNA-binding protein, protects pri-miRNAs from SERRATE-dependent exoribonuclease activities.

MAC5 is a component of the conserved MOS4-associated complex. It plays critical roles in development and immunity. Here we report that MAC5 is required for microRNA (miRNA) biogenesis. MAC5 interacts with Serrate (SE), which is a core component of the microprocessor that processes primary miRNA transcripts (pri-miRNAs) into miRNAs and binds the stem-loop region of pri-miRNAs. MAC5 is essential for both the efficient processing and the stability of pri-miRNAs. Interestingly, the reduction of pri-miRNA levels in mac5 is partially caused by XRN2/XRN3, the nuclear-localized 5'-to-3' exoribonucleases, and depends on SE. These results reveal that MAC5 plays a dual role in promoting pri-miRNA processing and stability through its interaction with SE and/or pri-miRNAs. This study also uncovers that pri-miRNAs need to be protected from nuclear RNA decay machinery, which is connected to the microprocessor.

Regulatory miPEP Open Reading Frames Contained in the Primary Transcripts of microRNAs.

This review aims to consider retrospectively the available data on the coding properties of pri-microRNAs and the regulatory functions of their open reading frames (ORFs) and the encoded peptides (miPEPs). Studies identifying miPEPs and analyzing the fine molecular mechanisms of their functional activities are reviewed together with a brief description of the methods to identify pri-miRNA ORFs and the encoded protein products. Generally, miPEPs have been identified in many plant species of several families and in a few animal species. Importantly, molecular mechanisms of the miPEP action are often quite different between flowering plants and metazoan species. Requirement for the additional studies in these directions is highlighted by alternative findings concerning negative or positive regulation of pri-miRNA/miRNA expression by miPEPs in plants and animals. Additionally, the question of how miPEPs are distributed in non-flowering plant taxa is very important for understanding the evolutionary origin of such micropeptides. Evidently, further extensive studies are needed to explore the functions of miPEPs and the corresponding ORFs and to understand the full set of their roles in eukaryotic organisms. Thus, we address the most recent integrative views of different genomic, physiological, and molecular aspects concerning the expression of miPEPs and their possible fine functions.

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.

In silico study predicts a key role of RNA-binding domains 3 and 4 in nucleolin-miRNA interactions.

RNA binding proteins (RBPs) regulate many important cellular processes through their interactions with RNA molecules. RBPs are critical for posttranscriptional mechanisms keeping gene regulation in a fine equilibrium. Conversely, dysregulation of RBPs and RNA metabolism pathways is an established hallmark of tumorigenesis. Human nucleolin (NCL) is a multifunctional RBP that interacts with different types of RNA molecules, in part through its four RNA binding domains (RBDs). Particularly, NCL interacts directly with microRNAs (miRNAs) and is involved in their aberrant processing linked with many cancers, including breast cancer. Nonetheless, molecular details of the NCL-miRNA interaction remain obscure. In this study, we used an in silico approach to characterize how NCL targets miRNAs and whether this specificity is imposed by a definite RBD-interface. Here, we present structural models of NCL-RBDs and miRNAs, as well as predict scenarios of NCL-miRNA interactions generated using docking algorithms. Our study suggests a predominant role of NCL RBDs 3 and 4 (RBD3-4) in miRNA binding. We provide detailed analyses of specific motifs/residues at the NCL-substrate interface in both these RBDs and miRNAs. Finally, we propose that the evolutionary emergence of more than two RBDs in NCL in higher organisms coincides with its additional role/s in miRNA processing. Our study shows that RBD3-4 display sequence/structural determinants to specifically recognize miRNA precursor molecules. Moreover, the insights from this study can ultimately support the design of novel antineoplastic drugs aimed at regulating NCL-dependent biological pathways with a causal role in tumorigenesis.