pri-miRNA Processing Research Articles

The structural landscape of Microprocessor-mediated processing of pri-let-7 miRNAs

MicroRNA (miRNA) biogenesis is initiated upon cleavage of a primary miRNA (pri-miRNA) hairpin by the Microprocessor (MP), composed of the Drosha RNase III enzyme and its partner DGCR8. Multiple pri-miRNA sequence motifs affect MP recognition, fidelity, and efficiency. Here, we performed cryoelectron microscopy (cryo-EM) and biochemical studies of several let-7 family pri-miRNAs in complex with human MP. We show that MP has the structural plasticity to accommodate a range of pri-miRNAs. These structures revealed key features of the 5' UG sequence motif, more comprehensively represented as the "flipped U with paired N"(fUN) motif. Our analysis explains how cleavage of class-II pri-let-7 members harboring a bulged nucleotide generates a non-canonical precursor with a 1-nt 3' overhang. Finally, the MP-SRSF3-pri-let-7f1 structure reveals how SRSF3 contributes to MP fidelity by interacting with the CNNC motif and Drosha's Piwi/Argonaute/Zwille (PAZ)-like domain. Overall, this study sheds light on the mechanisms for flexible recognition, accurate cleavage, and regulated processing of different pri-miRNAs by MP.

The structural landscape of Microprocessor mediated pri-let-7 miRNA processing.

miRNA biogenesis is initiated upon cleavage of a primary miRNA (pri-miRNA) hairpin by the Microprocessor (MP), composed of the Drosha RNase III enzyme and its partner DGCR8. Multiple pri-miRNA sequence motifs affect MP recognition, fidelity, and efficiency. Here, we performed cryo-EM and biochemical studies of several let-7 family pri-miRNAs in complex with human MP. We show that MP has the structural plasticity to accommodate a range of pri-miRNAs. These structures revealed key features of the 5' UG sequence motif, more comprehensively represented as the "fUN" motif. Our analysis explains how cleavage of class-II pri-let-7 members harboring a bulged nucleotide generates a noncanonical precursor with a 1-nt 3' overhang. Finally, the MP-SRSF3-pri-let-7f1 structure reveals how SRSF3 contributes to MP fidelity by interacting with the CNNC-motif and Drosha's PAZ-like domain. Overall, this study sheds light on the mechanisms for flexible recognition, accurate cleavage, and regulated processing of different pri-miRNAs by MP.

The spliceosome-associated protein CWC15 promotes miRNA biogenesis in Arabidopsis

MicroRNAs (miRNAs) play a key role in regulating gene expression and their biogenesis is precisely controlled through modulating the activity of microprocessor. Here, we report that CWC15, a spliceosome-associated protein, acts as a positive regulator of miRNA biogenesis. CWC15 binds the promoters of genes encoding miRNAs (MIRs), promotes their activity, and increases the occupancy of DNA-dependent RNA polymerases at MIR promoters, suggesting that CWC15 positively regulates the transcription of primary miRNA transcripts (pri-miRNAs). In addition, CWC15 interacts with Serrate (SE) and HYL1, two key components of microprocessor, and is required for efficient pri-miRNA processing and the HYL1-pri-miRNA interaction. Moreover, CWC15 interacts with the 20 S proteasome and PRP4KA, facilitating SE phosphorylation by PRP4KA, and subsequent non-functional SE degradation by the 20 S proteasome. These data reveal that CWC15 ensures optimal miRNA biogenesis by maintaining proper SE levels and by modulating pri-miRNA levels. Taken together, this study uncovers the role of a conserved splicing-related protein in miRNA biogenesis.

Structural atlas of human primary microRNAs generated by SHAPE-MaP

MicroRNA (miRNA) maturation is critically dependent on structural features of primary transcripts (pri-miRNAs). However, the scarcity of determined pri-miRNA structures has limited our understanding of miRNA maturation. Here, we employed selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP), a high-throughput RNA structure probing method, to unravel the secondary structures of 476 high-confidence human pri-miRNAs. Our SHAPE-based structures diverge substantially from those inferred solely from computation, particularly in the apical loop and basal segments, underlining the need for experimental data in RNA structure prediction. By comparing the structures with high-throughput processing data, we determined the optimal structural features of pri-miRNAs. The sequence determinants are influenced substantially by their structural contexts. Moreover, we identified an element termed the bulged GWG motif (bGWG) with a 3' bulge in the lower stem, which promotes processing. Our structure-function mapping better annotates the determinants of pri-miRNA processing and offers practical implications for designing small hairpin RNAs and predicting the impacts of miRNA mutations.

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.

The DEAD-box helicase RCF1 plays roles in miRNA biogenesis and RNA splicing in Arabidopsis.

RCF1 is a highly conserved DEAD-box RNA helicase found in yeast, plants, and mammals. Studies about the functions of RCF1 in plants are limited. Here, we uncovered the functions of RCF1 in Arabidopsis thaliana as a player in pri-miRNA processing and splicing, as well as in pre-mRNA splicing. A mutant with miRNA biogenesis defects was isolated, and the defect was traced to a recessive point mutation in RCF1 (rcf1-4). We show that RCF1 promotes D-body formation and facilitates the interaction between pri-miRNAs and HYL1. Finally, we show that intron-containing pri-miRNAs and pre-mRNAs exhibit a global splicing defect in rcf1-4. Together, this work uncovers roles for RCF1 in miRNA biogenesis and RNA splicing in Arabidopsis.

Adenovirus VA RNAs impair maturation of primary microRNA.

Adenovirus expresses two non-coding virus-associated (VA) RNAs: VA I RNA and VA II RNA. Adenovirus-expressed VA RNAs interfere with the microRNA (miRNA) pathway by competing with precursor miRNAs. The processing pattern of primary miRNA (pri-miRNA) and factors to affect its processing are not exactly known when using adenovirus for the delivery of pri-miRNA. To observe pri-miRNA processing, plasmid construct encoding pri-miRNA was co-transfected with VA I/II RNA expression plasmid, or recombinant adenovirus encoding pri-miRNA was generated and infected. Levels of miRNAs, VA I RNA and VA II RNA were analyzed by a quantitative real-time PCR (RT-PCR). VA I-II full-length RNA was analyzed by a RT-PCR. RNA immunoprecipitation analysis to pull-down the VA I-II full-length RNA binding with Drosha was conducted with Drosha antibody. pri-miRNA was normally processed into mature miRNA when it was expressed in cells using plasmid. However, miRNA maturation was impaired when pri-miRNA was delivered and expressed using adenovirus. Of note, pri-miRNA processing was observed to be blocked by VA RNA expression. Such blocked processing could be recovered by introducing antisense RNA of VA RNA, anti-3'VA RNA. In addition, VA RNAs were transcribed into VA I-II full-length RNA, which was found to bind and sequester Drosha. Adenovirus infection downregulated the processing of pri-miRNAs in cells, and such downregulation could be derived from VA I-II full-length RNAs in pri-miRNA-like form through competitively binding to Drosha protein. These results indicated that the expression of adenovirus VA RNAs should be inhibited for successful delivery and expression of pri-miRNA or shRNA in cells using adenovirus.

Modulation of miR-146b by N6-methyladenosine modification remodels tumor-associated macrophages and enhances anti-PD-1 therapy in colorectal cancer

PurposeMicroRNA-146b (miR-146b) alleviates experimental colitis in mice by mediating macrophage polarization and the release of inflammatory factors. Our goals were to evaluate the antitumor efficacy of miR-146b in colorectal cancer (CRC) and to investigate the underlying mechanisms.MethodsWe used murine models of CRC to evaluate whether miR-146b influenced the progression of tumors independent of tumor-associated macrophages (TAMs). RNA immunoprecipitation, N6-methyladenosine (m6A) RNA immunoprecipitation and in vitro pri-miRNA processing assays were conducted to examine whether m6A mediates the maturation of pri-miR-146b/miR-146b. In a series of in vitro and in vivo experiments, we further defined the molecular mechanisms of methyltransferase-like 3 (METTL3)/miR-146b-mediated antitumor immunity and its efficacy in combination with anti-PD-1 immunotherapy.ResultsWe found that miR-146b deletion supported tumor progression by increasing the number of alternatively activated (M2) TAMs. Mechanistically, the m6A-related “writer” protein METTL3 and “reader” protein HNRNPA2B1 controlled miR-146b maturation by regulating the m6A modification region of pri-miR-146b. Furthermore, miR-146b deletion promoted the polarization of M2-TAMs by enhancing phosphoinositide 3-kinase (PI3K)/AKT signaling, and this effect was mediated by the class IA PI3K catalytic subunit p110β, which reduced T cell infiltration, aggravated immunosuppression and ultimately promoted tumor progression. METTL3 knockdown or miR-146b deletion induced programmed death ligand 1 (PD-L1) production via the p110β/PI3K/AKT pathway in TAMs and consequently augmented the antitumor activity of anti-PD-1 immunotherapy.ConclusionsThe maturation of pri-miR-146b is m6A-dependent, and miR-146b deletion-mediated TAM differentiation promotes the development of CRC by activating the PI3K/AKT pathway, which induces upregulation of PD-L1 expression, inhibits T cell infiltration into the TME and enhances the antitumor activity of anti-PD-1 immunotherapy. The findings reveal that targeting miR-146b can serve as an adjuvant to anti-PD-1 immunotherapy.Graphical

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.

The HOS15-HDA9 complex associates with HYL1 to modulate miRNA expression in response to ABA signaling.

The regulation of microRNA (miRNA) biogenesis is crucial for maintaining plant homeostasis under biotic and abiotic stress. The crosstalk between the RNA polymerase II (Pol-II) complex and the miRNA processing machinery has emerged as a central hub modulating transcription and co-transcriptional processing of primary miRNA transcripts (pri-miRNAs). However, it remains unclear how miRNA-specific transcriptional regulators recognize MIRNA loci. Here, we show that the Arabidopsis (Arabidopsis thaliana) HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE15 (HOS15)-HISTONE DEACETYLASE9 (HDA9) complex is a conditional suppressor of miRNA biogenesis, particularly in response to ABA. When treated with ABA, hos15/hda9 mutants show enhanced transcription of pri-miRNAs that is accompanied by increased processing, leading to over-accumulation of a set of mature miRNAs. Moreover, upon recognition of the nascent pri-miRNAs, the ABA-induced recruitment of the HOS15-HDA9 complex to MIRNA loci is guided by HYPONASTIC LEAVES 1 (HYL1). The HYL1-dependent recruitment of the HOS15-HDA9 complex to MIRNA loci suppresses expression of MIRNAs and processing of pri-miRNA. Most importantly, our findings indicate that nascent pri-miRNAs serve as scaffolds for recruiting transcriptional regulators, specifically to MIRNA loci. This indicates that RNA molecules can act as regulators of their own expression by causing a negative feedback loop that turns off their transcription, providing a self-buffering system.

FTO-mediated m6A demethylation of pri-miR-3591 alleviates osteoarthritis progression

ObjectivesIncreasing evidence have demonstrated the N6-methyladenosine (m6A) plays critical roles in osteoarthritis (OA) progression, but the role of m6A in OA has not been completely illuminated. Herein, we investigated the function and underlying mechanism of m6A demethylase fat mass and obesity-associated protein (FTO) in OA progression.Materials and methodsThe FTO expression was detected in mice OA cartilage tissues and lipopolysaccharide (LPS)-stimulated chondrocytes. Gain-of-function assays was used to evaluate the role of FTO in OA cartilage injury in vitro and in vivo. The miRNA-sequencing, RNA-binding protein immunoprecipitation (RIP), luciferase reporter assay, and in vitro pri-miRNA processing assays were conducted to confirm that FTO modulated the pri-miR-3591 process in an m6A-dependent manner and then the binding sites of miR-3591-5p with PRKAA2.ResultsFTO was outstandingly downregulated in LPS-stimulated chondrocytes and OA cartilage tissues. FTO overexpression enhanced the proliferation, suppressed apoptosis, and decreased degradation of extracellular matrix in LPS-induced chondrocytes, whereas FTO knockdown contributed to the opposite effects. In vivo animal experiments showed that FTO overexpression markedly alleviated OA mice cartilage injury. Mechanically, FTO-mediated m6A demethylation of pri-miR-3591 leaded to a maturation block of miR-3591-5p, which relieved the inhibitory effect of miR-3591-5p on PRKAA2 and then promoted the increase of PRKAA2, thereby alleviating OA cartilage damage.ConclusionsOur results attested that FTO alleviated the OA cartilage damage by mediating FTO/miR-3591-5p/PRKAA2 axis, which provided fresh insights into the therapeutic strategies for OA.

Intrinsically disordered proteins SAID1/2 condensate on SERRATE for dual inhibition of miRNA biogenesis in Arabidopsis

Intrinsically disordered proteins (IDPs) SAID1/2 are hypothetic dentin sialophosphoprotein-like proteins, but their true functions are unknown. Here, we identified SAID1/2 as negative regulators of SERRATE (SE), a core factor in miRNA biogenesis complex (microprocessor). Loss-of-function double mutants of said1; said2 caused pleiotropic developmental defects and thousands of differentially expressed genes that partially overlapped with those in se. said1; said2 also displayed increased assembly of microprocessor and elevated accumulation of microRNAs (miRNAs). Mechanistically, SAID1/2 promote pre-mRNA processing 4 kinase A-mediated phosphorylation of SE, causing its degradation invivo. Unexpectedly, SAID1/2 have strong binding affinity to hairpin-structured pri-miRNAs and can sequester them from SE. Moreover, SAID1/2 directly inhibit pri-miRNA processing by microprocessor invitro. Whereas SAID1/2 did not impact SE subcellular compartmentation, the proteins themselves exhibited liquid-liquid phase condensation that is nucleated on SE. Thus, we propose that SAID1/2 reduce miRNA production through hijacking pri-miRNAs to prevent microprocessor activity while promoting SE phosphorylation and its destabilization in Arabidopsis.

MicroRNA production in Arabidopsis.

In plants, microRNAs (miRNAs) associate with ARGONAUTE (AGO) proteins and act as sequence-specific repressors of target gene expression, at the post-transcriptional level through target transcript cleavage and/or translational inhibition. MiRNAs are mainly transcribed by DNA-dependent RNA polymerase II (POL II) and processed by DICER LIKE1 (DCL1) complex into 21∼22 nucleotide (nt) long. Although the main molecular framework of miRNA biogenesis and modes of action have been established, there are still new requirements continually emerging in the recent years. The studies on the involvement factors in miRNA biogenesis indicate that miRNA biogenesis is not accomplished separately step by step, but is closely linked and dynamically regulated with each other. In this article, we will summarize the current knowledge on miRNA biogenesis, including MIR gene transcription, primary miRNA (pri-miRNA) processing, miRNA AGO1 loading and nuclear export; and miRNA metabolism including methylation, uridylation and turnover. We will describe how miRNAs are produced and how the different steps are regulated. We hope to raise awareness that the linkage between different steps and the subcellular regulation are becoming important for the understanding of plant miRNA biogenesis and modes of action.

Recent Insights into Plant miRNA Biogenesis: Multiple Layers of miRNA Level Regulation.

MicroRNAs are small RNAs, 20-22 nt long, the main role of which is to downregulate gene expression at the level of mRNAs. MiRNAs are fundamental regulators of plant growth and development in response to internal signals as well as in response to abiotic and biotic factors. Therefore, the deficiency or excess of individual miRNAs is detrimental to particular aspects of a plant's life. In consequence, the miRNA levels must be appropriately adjusted. To obtain proper expression of each miRNA, their biogenesis is controlled at multiple regulatory layers. Here, we addressed processes discovered to influence miRNA steady-state levels, such as MIR transcription, co-transcriptional pri-miRNA processing (including splicing, polyadenylation, microprocessor assembly and activity) and miRNA-encoded peptides synthesis. MiRNA stability, RISC formation and miRNA export out of the nucleus and out of the plant cell also define the levels of miRNAs in various plant tissues. Moreover, we show the evolutionary conservation of miRNA biogenesis core proteins across the plant kingdom.

Understanding the evolution of miRNA biogenesis machinery in plants with special focus on rice.

miRNA biogenesis process is an intricate and complex event consisting of many proteins working in a highly coordinated fashion. Most of these proteins have been studied in Arabidopsis; however, their orthologs and functions have not been explored in other plant species. In the present study, we have manually curated all the experimentally verified information present in the literature regarding these proteins and found a total of 98 genes involved in miRNA biogenesis in Arabidopsis. The conservation pattern of these proteins was identified in other plant species ranging from dicots to lower organisms, and we found that a major proportion of proteins involved in the pri-miRNA processing are conserved. However, nearly 20% of the genes, mostly involved in either transcription or functioning of the miRNAs, were absent in the lower organisms. Further, we manually curated a regulatory network of the core components of the biogenesis process and found that nearly half (46%) of the proteins interact with them, indicating that the processing step is perhaps the most under surveillance/regulation. We have subsequently attempted to characterize the orthologs identified in Oryza sativa, on the basis of transcriptome and epigenetic modifications under field drought conditions in order to assess the impact of drought on the process. We found several participating genes to be differentially expressed and/or epigenetically methylated under drought, although the core components like DCL1, SE, and HYL1 remain unaffected by the stress itself. The study enhances our present understanding of the biogenesis process and its regulation.

MicroRNA biogenesis pathway alterations in aging

Aging is characterized by genomic instability and dysregulation of gene expression. MicroRNAs (miRNAs) are small non-coding RNAs that play a crucial role in post-transcriptional gene regulation. This work explores the impact of dysregulated miRNA biogenesis on the aging process. During aging, alterations in the transcription of primary miRNAs (pri-miRNAs) occur due to genomic changes, DNA damage, and epigenetic modifications. The microprocessor complex, comprising DGCR8 and Drosha proteins, is vital for pri-miRNA processing. Age-related changes in this complex affect miRNA biogenesis and miRNA expression profiles, linking these alterations with age-related conditions. Conversely, interventions like caloric restriction and mTOR inhibition enhance microprocessor activity, suggesting a connection between microprocessor function, aging-related pathways, and lifespan extension. Exportin-5 mediates the transport of pre-miRNAs from the nucleus to the cytoplasm. Although the role of miRNA export in aging is not well understood, accelerated export of pre-miRNAs is observed in response to DNA damage, and nucleocytoplasmic transport has been linked to cellular senescence. Dicer is responsible for processing pre-miRNAs into mature miRNAs. Reduced Dicer expression during aging is reported in various organisms and tissues and is associated with premature aging phenotypes. Conversely, the upregulation of Dicer improves stress resistance and metabolic adaptations induced by caloric restriction and exercise training. Understanding the role of miRNA biogenesis disruption in aging provides insights into the molecular mechanisms of aging and age-related diseases. Targeting this pathway may hold promise for therapeutic strategies and contribute to healthy aging.

Abstract 11998: ADAR1 Mediates Macrophage Activation by Its Non-Editing Activity-Mediated Pri-miRNA Processing in Abdominal Aortic Aneurysm

Introduction: Macrophage activation plays a critical role in abdominal aortic aneurysm (AAA) development. However, molecular mechanisms controlling macrophage activation in AAA remain largely unknown. The objective of the study was to identify novel mechanisms underlying adenosine deaminase acting on RNA (ADAR1) function in macrophage activation and AAA formation. Methods: Aortic transplantation was conducted to determine the importance of non-vascular ADAR1 in AAA development. Angiotensin II (Ang II) infusion of ApoE–/– mouse combined with macrophage-specific knockout of ADAR1 was used to study ADAR1 macrophage-specific role in AAA formation. Primary cultured bone-marrow derived and peritoneal macrophages were used to study how ADAR1 regulates macrophage activation. Moreover, a novel humanized AAA model was established by transplanting human artery to immunodeficient mouse aorta followed by immunity reconstitution with ADAR1-deficient human monocytes to test the role of human macrophages in aneurysm formation in human arteries. Results: Allograft transplantation of wild type abdominal aortas to ADAR1+/- recipient mice significantly attenuated AAA formation, suggesting that non-vascular ADAR1 is essential for AAA development. ADAR1 deficiency in hematopoietic cells decreased the prevalence of AAA while inhibited macrophage infiltration and aorta wall inflammation. ADAR1 deletion blocked the classic macrophage activation, diminished NF-κB signaling, and enhanced the expression of many anti-inflammatory miRNAs. Mechanistically, ADAR1 interacted with Drosha to promote its degradation, which attenuated Drosha-DGCR8 interaction, and consequently inhibited pri- to pre-miRNA processing of miRNAs targeting IKKβ, resulting in an increased IKKβ expression and enhanced NF-κB signaling. Reconstitution of ADAR1-deficient, but not the wild type, human monocytes to immunodeficient mice blocked the aneurysm formation in transplanted human arteries. Conclusions: Macrophage ADAR1 promotes AAA formation in both mouse and human arteries through a novel mechanism, i.e., Drosha protein degradation, which inhibits the processing of miRNAs targeting NF-kB signaling and thus elicits macrophage-mediated vascular inflammation.

Partial Disturbance of Microprocessor Function in Human Stem Cells Carrying a Heterozygous Mutation in the DGCR8 Gene.

Maturation of microRNAs (miRNAs) begins by the "Microprocessor" complex, containing the Drosha endonuclease and its partner protein, "DiGeorge Syndrome Critical Region 8" (DGCR8). Although the main function of the two proteins is to coordinate the first step of precursor miRNAs formation, several studies revealed their miRNA-independent functions in other RNA-related pathways (e.g., in snoRNA decay) or, for the DGCR8, the role in tissue development. To investigate the specific roles of DGCR8 in various cellular pathways, we previously established a human embryonic stem-cell (hESC) line carrying a monoallelic DGCR8 mutation by using the CRISPR-Cas9 system. In this study, we genetically characterized single-cell originated progenies of the cell line and showed that DGCR8 heterozygous mutation results in only a modest effect on the mRNA level but a significant decrease at the protein level. Self-renewal and trilineage differentiation capacity of these hESCs were not affected by the mutation. However, partial disturbance of the Microprocessor function could be revealed in pri-miRNA processing along the human chromosome 19 miRNA cluster in several clones. With all these studies, we can demonstrate that the mutant hESC line is a good model to study not only miRNA-related but also other "noncanonical" functions of the DGCR8 protein.

Implication of genetic variants in primary microRNA processing sites in the risk of multiple sclerosis.

SummaryBackgroundMultiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system with a well-established genetic contribution to susceptibility. Over 200 genetic regions have been linked to the inherited risk of developing MS, but the disease-causing variants and their functional effects at the molecular level are still largely unresolved. We hypothesised that MS-associated single-nucleotide polymorphisms (SNPs) affect the recognition and enzymatic cleavage of primary microRNAs (pri-miRNAs).MethodsOur study focused on 11 pri-miRNAs (9 primate-specific) that are encoded in genetic risk loci for MS. The levels of mature miRNAs and potential isoforms (isomiRs) produced from those pri-miRNAs were measured in B cells obtained from the peripheral blood of 63 MS patients and 28 healthy controls. We tested for associations between SNP genotypes and miRNA expression in cis using quantitative trait locus (cis-miR-eQTL) analyses. Genetic effects on miRNA stem-loop processing efficiency were verified using luciferase reporter assays. Potential direct miRNA target genes were identified by transcriptome profiling and computational binding site assessment.FindingsMature miRNAs and isomiRs from hsa-mir-26a-2, hsa-mir-199a-1, hsa-mir-4304, hsa-mir-4423, hsa-mir-4464 and hsa-mir-4492 could be detected in all B-cell samples. When MS patient subgroups were compared with healthy controls, a significant differential expression was observed for miRNAs from the 5’ and 3’ strands of hsa-mir-26a-2 and hsa-mir-199a-1. The cis-miR-eQTL analyses and reporter assays pointed to a slightly more efficient Drosha-mediated processing of hsa-mir-199a-1 when the MS risk allele T of SNP rs1005039 is present. On the other hand, the MS risk allele A of SNP rs817478, which substitutes the first C in a CNNC sequence motif, was found to cause a markedly lower efficiency in the processing of hsa-mir-4423. Overexpression of hsa-mir-199a-1 inhibited the expression of 60 protein-coding genes, including IRAK2, MIF, TNFRSF12A and TRAF1. The only target gene identified for hsa-mir-4423 was TMEM47.InterpretationWe found that MS-associated SNPs in sequence determinants of pri-miRNA processing can affect the expression of mature miRNAs. Our findings complement the existing literature on the dysregulation of miRNAs in MS. Further studies on the maturation and function of miRNAs in different cell types and tissues may help to gain a more detailed functional understanding of the genetic basis of MS.FundingThis study was funded by the Rostock University Medical Center (FORUN program, grant: 889002), Sanofi Genzyme (grant: GZ-2016-11560) and Merck Serono GmbH (Darmstadt, Germany, an affiliate of Merck KGaA, CrossRef Funder ID: 10.13039/100009945, grant: 4501860307). NB was supported by the Stiftung der Deutschen Wirtschaft (sdw) and the FAZIT foundation. EP was supported by the Landesgraduiertenförderung Mecklenburg-Vorpommern.

Nucleolin’s Role in the Biogenesis of Pri‐miRNA 16 by the Microprocessor Complex: An In silico Approach

MicroRNAs (miRNAs) are short, non‐coding strands of RNA found in eukaryotes. They play a role in gene silencing through inhibiting translation or degrading mRNA. Primary miRNA (pri‐miRNA) processing is the initial step in producing a mature microRNA molecule. The microprocessor complex (MP) proteins, DROSHA and DGCR8, cleaves the pri‐miRNA molecule to a shorter hairpin structure. This cleaved RNA hairpin, precursor miRNA, is sent to the cytoplasm for further processing into the mature miRNA. Previous research has uncovered that the expression and cellular localization of the stress‐responsive protein, nucleolin (NCL) affects the processing of several miRNAs, including microRNA 16 (miR16). Nucleolin is an RNA binding protein and is primarily located in the nucleolus but can also be localized in various cellular compartments. When NCL is present in the nucleus, the MP complex works more efficiently in cleaving pri‐miR 16‐1 to its pre‐miRNA form resulting in an upregulation of miR 16‐1. It has been proposed that nucleolin binds to pri‐miR 16‐1 and stabilizes its confirmation for cleavage by the MP complex. However, the mechanistic and structural details on how nucleolin interacts with pri‐miRNA 16‐1 or the MP are still unclear. It is also unknown if NCL is similarly involved in miR 16‐2 biogenesis. NCL and miRNA 16 are also intricately involved in the regulation of the expression of bcl2, an anti‐apoptotic and oncogenic protein. When NCL is in the cytoplasm, it stabilizes the 3’ untranslated region of bcl2 mRNA leading to an increased expression of the protein. Unlike nucleolin, miR 16 represses the translation of bcl2 mRNA. At high levels of miR 16 when NCL is localized to the nucleus, bcl2 protein expression is low. When cellular stress occurs, NCL returns to the cytoplasm to stabilize bcl2 and this also results in less efficiency in pri‐miR 16 processing, resulting in an overall increase in bcl2 protein. It is evident that NCL balances the levels of bcl2 and miR16 through its cellular localization. Since the molecular mechanism underlying NCL’s role in the MP‐based processing of pri‐miR16‐1 remains largely unknown, in this study, we used computational approaches including structural alignments, protein‐protein, and protein‐RNA docking analyses to determine how pri‐miR 16‐1, nucleolin, and the MP complex interact. In addition, we have investigated if pri‐miR 16‐2 interacts with the NCL and the MP complex in a similar fashion. We show in our predictive models that the interaction interface of pri‐miR 16‐1 and pri‐miR 16‐2 with nucleolin and the MP complex nucleolin is distinct. For pri‐miR 16‐1, the interaction interface involves NCL and DGCR8 B and C, but in the case of pri‐miR 16‐2, it involves NCL and DROSHA. Additionally, in the presence of nucleolin, the hydrogen bond interactions are reduced between pri‐miR 16‐2 and the MP complex. Although the number of interactions between the different components of the MP complex is the same in both scenarios, the specific residues that form the interaction interface differ, suggesting distinct mechanisms. Since the levels of miR16 expression can lead to apoptosis of cancerous cells and prevent cell proliferation, the results of our study provide important clues in understanding how the NCL‐miR‐MP complex can be targeted in drug design for cancer therapy.