Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a.

The CRISPR-associated protein Cas12a (Cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide RNAs and RNA-guided DNase activity for target DNA cleavage. To elucidate the molecular basis of both activities, we determined crystal structures of Francisella novicida Cas12a bound to guide RNA and in complex with an R-loop formed by a non-cleavable guide RNA precursor and a full-length target DNA. Corroborated by biochemical experiments, these structures reveal the mechanisms of guide RNA processing and pre-ordering of the seed sequence in the guide RNA that primes Cas12a for target DNA binding. Furthermore, the R-loop complex structure reveals the strand displacement mechanism that facilitates guide-target hybridization and suggests a mechanism for double-stranded DNA cleavage involving a single active site. Together, these insights advance our mechanistic understanding of Cas12a enzymes and may contribute to further development of genome editing technologies.

Regulatory network of miRNA on its target: coordination between transcriptional and post-transcriptional regulation of gene expression.

MicroRNAs (miRNAs) are a class of endogenous small noncoding RNAs that participate in a majority of biological processes via regulating target gene expression. The post-transcriptional repression through miRNA seed region binding to 3' UTR of target mRNA is considered as the canonical mode of miRNA-mediated gene regulation. However, emerging evidence suggests that other regulatory modes exist beyond the canonical mechanism. In particular, the function of intranuclear miRNA in gene transcriptional regulation is gradually revealed, with evidence showing their contribution to gene silencing or activating. Therefore, miRNA-mediated regulation of gene transcription not only expands our understanding of the molecular mechanism underlying miRNA regulatory function, but also provides new evidence to explain its ability in the sophisticated regulation of many bioprocesses. In this review, mechanisms of miRNA-mediated gene transcriptional and post-transcriptional regulation are summarized, and the synergistic effects among these actions which form a regulatory network of a miRNA on its target are particularly elaborated. With these discussions, we aim to emphasize the importance of miRNA regulatory network on target gene regulation and further highlight the potential application of the network mode in the achievement of a more effective and stable modulation of the target gene expression.

Defining the seed sequence of the Cas12b CRISPR-Cas effector complex.

Target binding by CRISPR-Cas ribonucleoprotein effectors is initiated by the recognition of double-stranded PAM motifs by the Cas protein moiety followed by destabilization, localized melting, and interrogation of the target by the guide part of CRISPR RNA moiety. The latter process depends on seed sequences, parts of the target that must be strictly complementary to CRISPR RNA guide. Mismatches between the target and CRISPR RNA guide outside the seed have minor effects on target binding, thus contributing to off-target activity of CRISPR-Cas effectors. Here, we define the seed sequence of the Type V Cas12b effector from Bacillus thermoamylovorans. While the Cas12b seed is just five bases long, in contrast to all other effectors characterized to date, the nucleotide base at the site of target cleavage makes a very strong contribution to target binding. The generality of this additional requirement was confirmed during analysis of target recognition by Cas12b effector from Alicyclobacillus acidoterrestris. Thus, while the short seed may contribute to Cas12b promiscuity, the additional specificity determinant at the site of cleavage may have a compensatory effect making Cas12b suitable for specialized genome editing applications.

A seed motif for target RNA capture enables efficient immune defence by a type III-B CRISPR-Cas system.

CRISPR-Cas systems provide an adaptive defence against foreign nucleic acids guided by small RNAs (crRNAs) in archaea and bacteria. The Type III CRISPR systems are reported to carry RNase, RNA-activated DNase and cyclic oligoadenylate (cOA) synthetase activity, and are significantly different from other CRISPR systems. However, detailed features of target recognition, which are essential for enhancing target specificity remain unknown in Type III CRISPR systems. Here, we show that the Type III-B Cmr-α system in S. islandicus generates two constant lengths of crRNA independent of the length of the spacer. Either mutation at the 3'-end of crRNA or target truncation greatly influences the target capture and cleavage by the Cmr-α effector complex. Furthermore, we found that cleavage at the tag-proximal site on the target RNA by the Cmr-α RNP complex is delayed relative to the other sites, which probably provides Cas10 more time to function as a guard against invaders. Using a mutagenesis assay in vivo, we discovered that a seed motif located at the tag-distal region of the crRNA is required by Cmr1α for target RNA capture by the Cmr-α system thereby enhancing target specificity and efficiency. These findings further refine the model for immune defence of Type III-B CRISPR-Cas system, commencing on capture, cleavage and regulation.

ImiRP: a computational approach to microRNA target site mutation.

BACKGROUND: MicroRNAs (miRNAs) are small ~22 nucleotide non-coding RNAs that function as post-transcriptional regulators of messenger RNA (mRNA) through base-pairing to 6-8 nucleotide long target sites, usually located within the mRNA 3' untranslated region. A common approach to validate and probe microRNA-mRNA interactions is to mutate predicted target sites within the mRNA and determine whether it affects miRNA-mediated activity. The introduction of miRNA target site mutations, however, is potentially problematic as it may generate new, "illegitimate sites" target sites for other miRNAs, which may affect the experimental outcome. While it is possible to manually generate and check single miRNA target site mutations, this process can be time consuming, and becomes particularly onerous and error prone when multiple sites are to be mutated simultaneously. We have developed a modular Java-based system called ImiRP (Illegitimate miRNA Predictor) to solve this problem and to facilitate miRNA target site mutagenesis. RESULTS: The ImiRP interface allows users to input a sequence of interest, specify the locations of multiple predicted target sites to mutate, and set parameters such as species, mutation strategy, and disallowed illegitimate target site types. As mutant sequences are generated, ImiRP utilizes the miRBase high confidence miRNA dataset to identify illegitimate target sites in each mutant sequence by comparing target site predictions between input and mutant sequences. ImiRP then assembles a final mutant sequence in which all specified target sites have been mutated. CONCLUSIONS: ImiRP is a mutation generator program that enables selective disruption of specified miRNA target sites while ensuring predicted target sites for other miRNAs are not inadvertently created. ImiRP supports mutagenesis of single and multiple miRNA target sites within a given sequence, including sites that overlap. This software will be particularly useful for studies looking at microRNA cooperativity, where mutagenesis of multiple microRNA target sites may be desired. The software is available at imirp.org and is available open source for download through GitHub ( https://github.com/imirp ).

The highly expressed 5'isomiR of hsa-miR-140-3p contributes to the tumor-suppressive effects of miR-140 by reducing breast cancer proliferation and migration.

BACKGROUND: miRNAs are small noncoding RNA molecules that play an important role in post-transcriptional regulation of gene expression. Length and/or sequence variants of the same miRNA are termed isomiRs. While most isomiRs are functionally redundant compared to their canonical counterparts, the so-called 5'isomiRs exhibit a shifted 5' end and therefore a shifted seed sequence resulting in a different target spectrum. However, not much is known about the functional relevance of these isoforms. RESULTS: Analysis of miRNA-seq data from breast cancer cell lines identified six pairs of highly expressed miRNAs and associated 5'isomiRs. Among them, hsa-miR-140-3p was of particular interest because its 5'isomiR showed higher expression compared to the canonical miRNA annotated in miRbase. This miRNA has previously been shown to control stemness of breast cancer cells. miRNAseq data of breast cancer patients (TCGA dataset) showed that both the canonical hsa-miR-140-3p and its 5'isomiR-140-3p were highly expressed in patients' tumors compared to normal breast tissue. In the current work, we present the functional characterization of 5'isomiR-140-3p and the cellular phenotypes associated with its overexpression in MCF10A, MDA-MB-468 and MDA-MB-231 cell lines in comparison to the canonical hsa-miR-140-3p. Contrary to the effect of the canonical hsa-miR-140-3p, overexpression of the 5'isomiR-140-3p led to a decrease in cell viability. The latter observation was supported by cell cycle analysis, where the 5'isomiR-140-3p but not the hsa-miR-140-3p caused cell cycle arrest in G0/G1-phase. Additionally, 5'ismoiR-140-3p overexpression was found to cause a decrease in cell migration in the three cell lines. We identified three novel direct target genes of the 5'isomiR-140-3p; COL4A1, ITGA6 and MARCKSL1. Finally, we have shown that knocking down these genes partially phenocopied the effects of the 5'isomiR-140-4p overexpression, where COL4A1 and ITGA6 knockdown led to reduced cell viability and cell cycle arrest, while MARCKSL1 knockdown resulted in a decrease in the migratory potential of cells. CONCLUSIONS: In summary, this work presents evidence that there is functional synergy between the canonical hsa-miR-140-3p and the newly identified 5'isomiR-140-3p in suppressing growth and progression of breast cancer by simultaneously targeting genes related to differentiation, proliferation, and migration.

Systematic analysis of berberine-induced signaling pathway between miRNA clusters and mRNAs and identification of mir-99a ∼ 125b cluster function by seed-targeting inhibitors in multiple myeloma cells.

BACKGROUND: Berberine (BBR) is a natural alkaloid derived from a traditional Chinese herbal medicine. However, the exact mechanisms underlying the different effects of berberine on MM cells have not been fully elucidated. METHODS: A systematic analysis assay integrated common signaling pathways modulated by the 3 miRNA clusters and mRNAs in MM cells after BBR treatment. The role of the mir-99a ∼ 125b cluster, an important oncomir in MM, was identified by comparing the effects of t-anti-mirs with complete complementary antisense locked nucleic acids (LNAs) against mature mir-125b (anti-mir-125b). RESULTS: Three miRNAs clusters (miR-99a ∼ 125b, miR-17 ∼ 92 and miR-106 ∼ 25) were significantly down-regulated in BBR-treated MM cells and are involved in multiple cancer-related signaling pathways. Furthermore, the top 5 differentially regulated genes, RAC1, NFκB1, MYC, JUN and CCND1 might play key roles in the progression of MM. Systematic integration revealed that 3 common signaling pathways (TP53, Erb and MAPK) link the 3 miRNA clusters and the 5 key mRNAs. Meanwhile, both BBR and seed-targeting t-anti-mir-99a ∼ 125b cluster LNAs significantly induced apoptosis, G2-phase cell cycle arrest and colony inhibition. CONCLUSIONS: our results suggest that BBR suppresses multiple myeloma cells, partly by down-regulating the 3 miRNA clusters and many mRNAs, possibly through TP53, Erb and MAPK signaling pathways. The mir-99a ∼ 125b cluster might be a novel target for MM treatment. These findings provide new mechanistic insight into the anticancer effects of certain traditional Chinese herbal medicine compounds.

Complementarity to an miRNA seed region is sufficient to induce moderate repression of a target transcript in the unicellular green alga Chlamydomonas reinhardtii.

MicroRNAs (miRNAs) are 20-24 nt non-coding RNAs that play important regulatory roles in a broad range of eukaryotes by pairing with mRNAs to direct post-transcriptional repression. The mechanistic details of miRNA-mediated post-transcriptional regulation have been well documented in multicellular model organisms. However, this process remains poorly studied in algae such as Chlamydomonas reinhardtii, and specific features of miRNA biogenesis, target mRNA recognition and subsequent silencing are not well understood. In this study, we report on the characterization of a Chlamydomonas miRNA, cre-miR1174.2, which is processed from a near-perfect hairpin RNA. Using Gaussia luciferase (gluc) reporter genes, we have demonstrated that cre-miR1174.2 is functional in Chlamydomonas and capable of triggering site-specific cleavage at the center of a perfectly complementary target sequence. A mismatch tolerance test assay, based on pools of transgenic strains, revealed that target hybridization to nucleotides of the seed region, at the 5' end of an miRNA, was sufficient to induce moderate repression of expression. In contrast, pairing to the 3' region of the miRNA was not critical for silencing. Our results suggest that the base-pairing requirements for small RNA-mediated repression in C. reinhardtii are more similar to those of metazoans compared with the extensive complementarity that is typical of land plants. Individual Chlamydomonas miRNAs may potentially modulate the expression of numerous endogenous targets as a result of these relaxed base-pairing requirements.

Synchronous Mutual Learning Network and Asynchronous Multi-Scale Embedding Network for miRNA-Disease Association Prediction.

MicroRNA (miRNA) serves as a pivotal regulator of numerous cellular processes, and the identification of miRNA-disease associations (MDAs) is crucial for comprehending complex diseases. Recently, graph neural networks (GNN) have made significant advancements in MDA prediction. However, these methods tend to learn one type of node representation from a single heterogeneous network, ignoring the importance of multiple network topologies and node attributes. Here, we propose SMDAP (Sequence hierarchical modeling-based Mirna-Disease Association Prediction framework), a novel GNN-based framework that incorporates multiple network topologies and various node attributes including miRNA seed and full-length sequences to predict potential MDAs. Specifically, SMDAP consists of two types of MDA representation: following a heterogeneous pattern, we construct a transfer learning-like synchronous mutual learning network to learn the first MDA representation in conjunction with the miRNA seed sequence. Meanwhile, following a homogeneous pattern, we design a subgraph-inspired asynchronous multi-scale embedding network to obtain the second MDA representation based on the miRNA full-length sequence. Subsequently, an adaptive fusion approach is designed to combine the two branches such that we can score the MDAs by the downstream classifier and infer novel MDAs. Comprehensive experiments demonstrate that SMDAP integrates the advantages of multiple network topologies and node attributes into two branch representations. Moreover, the area under the receiver operating characteristic curve is 0.9622 on DB1, which is a 5.06% increase from the baselines. The area under the precision-recall curve is 0.9777, which is a 7.33% increase from the baselines. In addition, case studies on three human cancers validated the predictive performance of SMDAP. Overall, SMDAP represents a powerful tool for MDA prediction.

Essential requirements for the detection and degradation of invaders by the Haloferax volcanii CRISPR/Cas system I-B.

To fend off foreign genetic elements, prokaryotes have developed several defense systems. The most recently discovered defense system, CRISPR/Cas, is sequence-specific, adaptive and heritable. The two central components of this system are the Cas proteins and the CRISPR RNA. The latter consists of repeat sequences that are interspersed with spacer sequences. The CRISPR locus is transcribed into a precursor RNA that is subsequently processed into short crRNAs. CRISPR/Cas systems have been identified in bacteria and archaea, and data show that many variations of this system exist. We analyzed the requirements for a successful defense reaction in the halophilic archaeon Haloferax volcanii. Haloferax encodes a CRISPR/Cas system of the I-B subtype, about which very little is known. Analysis of the mature crRNAs revealed that they contain a spacer as their central element, which is preceded by an eight-nucleotide-long 5' handle that originates from the upstream repeat. The repeat sequences have the potential to fold into a minimal stem loop. Sequencing of the crRNA population indicated that not all of the spacers that are encoded by the three CRISPR loci are present in the same abundance. By challenging Haloferax with an invader plasmid, we demonstrated that the interaction of the crRNA with the invader DNA requires a 10-nucleotide-long seed sequence. In addition, we found that not all of the crRNAs from the three CRISPR loci are effective at triggering the degradation of invader plasmids. The interference does not seem to be influenced by the copy number of the invader plasmid.

An unbiased seed-based RNAi selection screen identifies small RNAs that inhibit androgen signaling and prostate cancer cell growth.

Blocking androgen receptor signaling is the mainstay of therapy for advanced prostate cancer (PCa). However, acquired resistance to single agents targeting this pathway results in the development of lethal castration-resistant PCa. Combination therapy approaches represent a promising strategy for the treatment of advanced disease. Here, we explore a therapeutic strategy for PCa based on the ability of shRNAs/siRNAs to function essentially as miRNAs and, via seed sequence complementarity, induce RNA interference of numerous targets simultaneously. We developed a library that contained shRNAs with all possible seed sequence combinations to identify those ones that most potently reduce cell growth and viability when expressed in PCa cells. Validation of some of these RNAi sequences indicated that the toxic effect is associated with seed sequence complementarity to the 3' UTR of AR coregulatory and essential genes. In fact, expression of siRNAs containing the identified toxic seed sequences led to global inhibition of AR-mediated gene expression and reduced expression of cell-cycle genes. When tested in mice, the toxic shRNAs also inhibited castration-resistant PCa and exhibited therapeutic efficacy in pre-established tumors. Our findings highlight RNAi of androgen signaling networks as a promising therapeutic strategy for PCa.

MicroRNA and Nonsense Transcripts as Putative Viral Evasion Mechanisms.

Viral proteins encode numerous antiviral activities to modify the host immunity. In this article, we hypothesize that viral genomes and gene transcripts interfere with host gene expression using passive mechanisms to deregulate host microRNA (miRNA) activity. We postulate that various RNA viruses mimic or block binding between a host miRNA and its target transcript, a phenomenon mediated by the miRNA seed site at the 5' end of miRNA. Virus-encoded miRNA seed sponges (vSSs) can potentially bind to host miRNA seed sites and prevent interaction with their native targets thereby relieving native miRNA suppression. In contrast, virus-encoded miRNA seed mimics (vSMs) may mediate considerable downregulation of host miRNA activity. We analyzed genomes from diverse RNA viruses for vSS and vSM signatures and found an abundance of these motifs indicating that this may be a mechanism of deceiving host immunity. Employing respiratory syncytial virus and measles virus as models, we reveal that regions surrounding vSS or vSM motifs have features characteristics of pre-miRNA templates and show that RSV viral transcripts are processed into small RNAs that may behave as vSS or vSM effectors. These data suggest that complex molecular interactions likely occur at the host-virus interface. Identifying the mechanisms in the network of interactions between the host and viral transcripts can help uncover ways to improve vaccine efficacy, therapeutics, and potentially mitigate the adverse events that may be associated with some vaccines.

State-of-the-Art on Viral microRNAs in HPV Infection and Cancer Development.

BACKGROUND: High-risk HPV subtypes are driving forces for human cancer development: HPV-16 and HPV-18 are responsible for most HPV-caused cancers. OBJECTIVE: This review describes the present knowledge on HR-HPV genomes coding potential for viral miRNAs. METHODS: HPV subtypes miRNA database, VIRmiRtar, has been constructed applying bioinformatics and a computational method, ViralMir, exploiting structural features, the presence of hairpins, and validation by comparison with RNA sequencing datasets. RESULTS: Several miRNA candidates have been localised in the genomes of high-risk HPV subtypes. Among these, HPV-16 miR-1, miR-2 and miR-3. The database contains a list of host candidate gene targets that may be responsible for the oncogenesis in the various cellular environments. CONCLUSION: miRNA silencing therapies, based on specific cellular uptake of miRNA mimics and antagomiRs, directed towards HPV encoded miRNAs and/or microRNAs deregulated in the host cells, could be a valuable approach to support pharmaceutical interventions in the treatment of HPV dependent cancers.

The Mechanism of Synchronous Precise Regulation of Two Shrimp White Spot Syndrome Virus Targets by a Viral MicroRNA.

MicroRNAs (miRNAs), important factors in animal innate immunity, suppress the expressions of their target genes by binding to target mRNA's 3' untranslated regions (3'UTRs). However, the mechanism of synchronous regulation of multiple targets by a single miRNA remains unclear. In this study, the interaction between a white spot syndrome virus (WSSV) miRNA (WSSV-miR-N32) and its two viral targets (wsv459 and wsv322) was characterized in WSSV-infected shrimp. The outcomes indicated that WSSV-encoded miRNA (WSSV-miR-N32) significantly inhibited virus infection by simultaneously targeting wsv459 and wsv322. The silencing of wsv459 or wsv322 by siRNA led to significant decrease of WSSV copies in shrimp, showing that the two viral genes were required for WSSV infection. WSSV-miR-N32 could mediate 5'-3' exonucleolytic digestion of its target mRNAs, which stopped at the sites of target mRNA 3'UTRs close to the sequence complementary to the miRNA seed sequence. The complementary bases (to the target mRNA sequence) of a miRNA 9th-18th non-seed sequence were essential for the miRNA targeting. Therefore, our findings presented novel insights into the mechanism of miRNA-mediated suppression of target gene expressions, which would be helpful for understanding the roles of miRNAs in innate immunity of invertebrate.

Human diseases caused by germline and somatic abnormalities in microRNA and microRNA-related genes.

The human genome harbors approximately 2000 genes that encode microRNAs (miRNAs), small non-coding RNAs of approximately 20-22 nt that mediate post-transcriptional gene silencing. MiRNAs are generated from long transcripts through stepwise processing by the Drosha/DGCR8, Exportin-5/RanGTP and Dicer/TRBP complexes. Given that the expression of each individual miRNA is tightly regulated, the altered expression of certain miRNAs plays a pivotal role in human diseases. For instance, germline and somatic mutations in the genes encoding the miRNA processing machinery have been reported in different cancers. Furthermore, certain miRNA genes are encoded within regions that are deleted or duplicated in individuals with chromosomal abnormalities, and the fact that the knockout of these miRNAs in animal models results in lethality or the abnormal development of certain tissues indicates that these miRNA genes contribute to the disease phenotypes. It has also been reported that mutations in miRNA genes or in miRNA-binding sites, which result in the impairment of tight regulation of target mRNA expression, cause human genetic diseases, although these cases are rare. This is in contrast to the aberrant expression of certain miRNAs that results from the impairment of transcriptional or post-transcriptional regulation, which has been reported frequently in various human diseases. The present review focuses on human diseases caused by mutations in genes encoding miRNAs and the miRNA processing machinery as well as in miRNA-binding sites. Furthermore, human diseases caused by chromosomal abnormalities that involve the deletion or duplication of regions harboring genes that encode miRNAs or the miRNA processing machinery are also introduced.