Structural and functional basis of mammalian microRNA biogenesis by Dicer.

MicroRNA (miRNA) and RNA interference (RNAi) pathways rely on small RNAs produced by Dicer endonucleases. Mammalian Dicer primarily supports the essential gene-regulating miRNA pathway, but how it is specifically adapted to miRNA biogenesis is unknown. We show that the adaptation entails a unique structural role of Dicer's DExD/H helicase domain. Although mice tolerate loss of its putative ATPase function, the complete absence of the domain is lethal because it assures high-fidelity miRNA biogenesis. Structures of murine Dicer•-miRNA precursor complexes revealed that the DExD/H domain has a helicase-unrelated structural function. It locks Dicer in a closed state, which facilitates miRNA precursor selection. Transition to a cleavage-competent open state is stimulated by Dicer-binding protein TARBP2. Absence of the DExD/H domain or its mutations unlocks the closed state, reduces substrate selectivity, and activates RNAi. Thus, the DExD/H domain structurally contributes to mammalian miRNA biogenesis and underlies mechanistical partitioning of miRNA and RNAi pathways.

Functional canonical RNAi in mice expressing a truncated Dicer isoform and long dsRNA.

Canonical RNA interference (RNAi) is sequence-specific mRNA degradation guided by small interfering RNAs (siRNAs) made by RNase III Dicer from long double-stranded RNA (dsRNA). RNAi roles include gene regulation, antiviral immunity or defense against transposable elements. In mammals, RNAi is constrained by Dicer's adaptation to produce another small RNA class-microRNAs. However, a truncated Dicer isoform (ΔHEL1) supporting RNAi exists in mouse oocytes. A homozygous mutation to express only the truncated ΔHEL1 variant causes dysregulation of microRNAs and perinatal lethality in mice. Here, we report the phenotype and canonical RNAi activity in DicerΔHEL1/wt mice, which are viable, show minimal miRNome changes, but their endogenous siRNA levels are an order of magnitude higher. We show that siRNA production in vivo is limited by available dsRNA, but not by Protein kinase R, a dsRNA sensor of innate immunity. dsRNA expression from a transgene yields sufficient siRNA levels to induce efficient RNAi in heart and muscle. DicerΔHEL1/wt mice with enhanced canonical RNAi offer a platform for examining potential and limits of mammalian RNAi in vivo.

The Effect of Alternative Splicing Sites on Mirtron Formation and Arm Selection of Precursor microRNAs.

Mirtrons represent a subclass of microRNAs (miRNAs) that rely on the splicing machinery for their maturation. However, the molecular details of this Drosha-independent processing are still not fully understood; as an example, the Microprocessor complex cannot process the mirtronic pre-miRNA from the transcript even if splice site mutations are present. To investigate the influence of alternative splicing sites on mirtron formation, we generated Enhanced Green Fluorescent Protein (EGFP) reporters containing artificial introns to compare the processing of canonical miRNAs and mirtrons. Although mutations of both splice sites generated a complex pattern of alternative transcripts, mirtron formation was always severely affected as opposed to the normal processing of the canonical hsa-mir-33b miRNA. However, we also detected that while its formation was also hindered, the mirtron-derived hsa-mir-877-3p miRNA was less affected by certain mutations than the hsa-mir-877-5p species. By knocking down Drosha, we showed that this phenomenon is not dependent on Microprocessor activity but rather points toward the potential stability difference between the miRNAs from the different arms. Our results indicate that when the major splice sites are mutated, mirtron formation cannot be rescued by nearby alternative splice sites, and stability differences between 5p and 3p species should also be considered for functional studies of mirtrons.

Comparing miRNA structure of mirtrons and non-mirtrons.

BACKGROUND: MicroRNAs proceeds through the different canonical and non-canonical pathways; the most frequent of the non-canonical ones is the splicing-dependent biogenesis of mirtrons. We compare the mirtrons and non-mirtrons of human and mouse to explore how their maturation appears in the precursor structure around the miRNA. RESULTS: We found the coherence of the overhang lengths what indicates the dependence between the cleavage sites. To explain this dependence we suggest the 2-lever model of the Dicer structure that couples the imprecisions in Drosha and Dicer. Considering the secondary structure of all animal pre-miRNAs we confirmed that single-stranded nucleotides tend to be located near the miRNA boundaries and in its center and are characterized by a higher mutation rate. The 5' end of the canonical 5' miRNA approaches the nearest single-stranded nucleotides what suggests the extension of the loop-counting rule from the Dicer to the Drosha cleavage site. A typical structure of the annotated mirtron pre-miRNAs differs from the canonical pre-miRNA structure and possesses the 1- and 2 nt hanging ends at the hairpin base. Together with the excessive variability of the mirtron Dicer cleavage site (that could be partially explained by guanine at its ends inherited from splicing) this is one more evidence for the 2-lever model. In contrast with the canonical miRNAs the mirtrons have higher snp densities and their pre-miRNAs are inversely associated with diseases. Therefore we supported the view that mirtrons are under positive selection while canonical miRNAs are under negative one and we suggested that mirtrons are an intrinsic source of silencing variability which produces the disease-promoting variants. Finally, we considered the interference of the pre-miRNA structure and the U2snRNA:pre-mRNA basepairing. We analyzed the location of the branchpoints and found that mirtron structure tends to expose the branchpoint site what suggests that the mirtrons can readily evolve from occasional hairpins in the immediate neighbourhood of the 3' splice site. CONCLUSION: The miRNA biogenesis manifests itself in the footprints of the secondary structure. Close inspection of these structural properties can help to uncover new pathways of miRNA biogenesis and to refine the known miRNA data, in particular, new non-canonical miRNAs may be predicted or the known miRNAs can be re-classified.

Alternative splicing of a viral mirtron differentially affects the expression of other microRNAs from its cluster and of the host transcript.

Interplay between alternative splicing and the Microprocessor may have differential effects on the expression of intronic miRNAs organized into clusters. We used a viral model - the LAT long non-coding RNA (LAT lncRNA) of Marek's disease oncogenic herpesvirus (MDV-1), which has the mdv1-miR-M8-M6-M7-M10 cluster embedded in its first intron - to assess the impact of splicing modifications on the biogenesis of each of the miRNAs from the cluster. Drosha silencing and alternative splicing of an extended exon 2 of the LAT lncRNA from a newly identified 3' splice site (SS) at the end of the second miRNA of the cluster showed that mdv1-miR-M6 was a 5'-tailed mirtron. We have thus identified the first 5'-tailed mirtron within a cluster of miRNAs for which alternative splicing is directly associated with differential expression of the other miRNAs of the cluster, with an increase in intronic mdv1-miR-M8 expression and a decrease in expression of the exonic mdv1-miR-M7, and indirectly associated with regulation of the host transcript. According to the alternative 3SS used for the host intron splicing, the mdv1-miR-M6 is processed as a mirtron by the spliceosome, dispatching the other miRNAs of the cluster into intron and exon, or as a canonical miRNA by the Microprocessor complex. The viral mdv1-miR-M6 mirtron is the first mirtron described that can also follow the canonical pathway.

Versatile microRNA biogenesis in animals and their viruses.

MicroRNAs (miRNAs) are ubiquitous gene regulators that modulate essential cellular processes at the post-transcriptional level. In metazoans and their viruses, most miRNAs are produced from hairpin-containing primary transcripts that are sequentially cleaved by nuclear Drosha and cytoplasmic Dicer. In the last decade, alternative mechanisms that bypass either the Drosha or Dicer cleavage step have emerged, increasing the complexity of the miRNA regulatory network. Here, we highlight non-canonical pathways that generate miRNAs using a variety of molecular machineries that play fundamental roles in the biogenesis and processing of other classes of cellular RNAs.

Promiscuous splicing-derived hairpins are dominant substrates of tailing-mediated defense of miRNA biogenesis in mammals.

Canonical microRNA (miRNA) hairpins are processed by the RNase III enzymes Drosha and Dicer into ∼22 nt RNAs loaded into an Argonaute (Ago) effector. In addition, splicing generates numerous intronic hairpins that bypass Drosha (mirtrons) to yield mature miRNAs. Here, we identify hundreds of previously unannotated, splicing-derived hairpins in intermediate-length (∼50-100 nt) but not small (20-30 nt) RNA data. Since we originally defined mirtrons from small RNA duplexes, we term this larger set as structured splicing-derived RNAs (ssdRNAs). These associate with Dicer and/or Ago complexes, but generally accumulate modestly and are poorly conserved. We propose they contaminate the canonical miRNA pathway, which consequently requires defense against the siege of splicing-derived substrates. Accordingly, ssdRNAs/mirtrons comprise dominant hairpin substrates for 3' tailing by multiple terminal nucleotidyltransferases, notably TUT4/7 and TENT2. Overall, the rampant proliferation of young mammalian mirtrons/ssdRNAs, coupled with an inhibitory molecular defense, comprises a Red Queen's race of intragenomic conflict.

Biogenesis, characterization, and functions of mirtrons.

MicroRNAs (miRNAs) are major post-transcriptional regulators of gene expression. They base pair with the complementary target mRNA at the 3'UTR and modulate cellular processes by repressing the mRNA translation or degrading the mRNA. There are well-documented mechanisms of biogenesis of miRNA; however, a sizeable number of miRNAs are also produced by non-canonical pathways. Mirtrons represent a predominant class of non-canonical miRNAs. Mirtrons originate from intronic regions and are produced in a splicing-dependent and Drosha-independent manner. Mirtrons constitute about 15% of all miRNAs produced in a human body and have caught attention of researchers worldwide due to their unconventional origin, sequence characteristics, evolutionary dynamics, ability to regulate variety of cellular processes and their immense potential in disease therapeutics. In this comprehensive review we collate the research done in the past decade including biogenesis, sequence characteristics, regulation, and emerging therapeutic roles of mirtrons. This article is categorized under: RNA Processing > Processing of Small RNAs Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs Regulatory RNAs/RNAi/Riboswitches > RNAi: Mechanisms of Action.

Posttranscriptional Regulation of the Human ABCG2 Multidrug Transporter Protein by Artificial Mirtrons.

ABCG2 is a membrane transporter protein that has been associated with multidrug resistance phenotype and tumor development. Additionally, it is expressed in various stem cells, providing cellular protection against endobiotics and xenobiotics. In this study, we designed artificial mirtrons to regulate ABCG2 expression posttranscriptionally. Applying EGFP as a host gene, we could achieve efficient silencing not only in luciferase reporter systems but also at the ABCG2 protein level. Moreover, we observed important new sequential-functional features of the designed mirtrons. Mismatch at the first position of the mirtron-derived small RNA resulted in better silencing than full complementarity, while the investigated middle and 3' mismatches did not enhance silencing. These latter small RNAs operated most probably via non-seed specific translational inhibition in luciferase assays. Additionally, we found that a mismatch in the first position has not, but a second mismatch in the third position has abolished target mRNA decay. Besides, one nucleotide mismatch in the seed region did not impair efficient silencing at the protein level, providing the possibility to silence targets carrying single nucleotide polymorphisms or mutations. Taken together, we believe that apart from establishing an efficient ABCG2 silencing system, our designing pipeline and results on sequential-functional features are beneficial for developing artificial mirtrons for other targets.

Dbr1 functions in mRNA processing, intron turnover and human diseases.

Pre-mRNA processing and mRNA stability play direct roles in controlling protein abundance in a cell. Before the mRNA can be translated into a protein, the introns in the pre-mRNA transcripts need to be removed by splicing, such that exons can be ligated together and can code for a protein. In this process, the function of the RNA lariat debranching enzyme or Dbr1 provides a rate-limiting step in the intron turnover process and possibly regulating the production of translation competent mRNAs. Surprising new roles of Dbr1 are emerging in cellular metabolism which extends beyond intron turnover processes, ranging from splicing regulation to translational control. In this review, we highlight the importance of the Dbr1 enzyme, its structure and how anomalies in its function could relate to various human diseases.

Two MicroRNAs Are Sufficient for Embryonic Patterning in C. elegans.

MicroRNAs (miRNAs) are a class of post-transcriptional repressors with diverse roles in animal development and physiology [1]. The Microprocessor complex, composed of Drosha and Pasha/DGCR8, is necessary for the biogenesis of all canonical miRNAs and essential for the early stages of animal embryogenesis [2-8]. However, the cause for this requirement is largely unknown. Animals often express hundreds of miRNAs, and it remains unclear whether the Microprocessor is required to produce one or few essential miRNAs or many individually non-essential miRNAs. Additionally, both Drosha and Pasha/DGCR8 bind and cleave a variety of non-miRNA substrates [9-15], and it is unknown whether these activities account for the Microprocessor's essential requirement. To distinguish between these possibilities, we developed a system in C. elegans to stringently deplete embryos of Microprocessor activity. Using a combination of auxin-inducible degradation (AID) and RNA interference (RNAi), we achieved Drosha and Pasha/DGCR8 depletion starting in the maternal germline, resulting in Microprocessor and miRNA-depleted embryos, which fail to undergo morphogenesis or form organs. Using a Microprocessor-bypass strategy, we show that this early embryonic arrest is rescued by the addition of just two miRNAs, one miR-35 and one miR-51 family member, resulting in morphologically normal larvae. Thus, just two out of ∼150 canonical miRNAs are sufficient for morphogenesis and organogenesis, and the processing of these miRNAs accounts for the essential requirement for Drosha and Pasha/DGCR8 during the early stages of C. elegans embryonic development. VIDEO ABSTRACT.

Detection and Verification of Mammalian Mirtrons by Northern Blotting.

microRNAs (miRNAs) have vital roles in regulating gene expression-contributing to major diseases like cancer and heart disease. Over the last decade, thousands of miRNAs have been discovered through high throughput sequencing-based annotation. Different classes have been described, as well as a great dynamic range of expression levels. While sequencing approaches provide insight into biogenesis and allow confident identification, there is a need for additional methods for validation and characterization. Northern blotting was one of the first techniques used for studying miRNAs, and remains one of the most valuable as it avoids enzymatic manipulation of miRNA transcripts. Blotting can also provide insight into biogenesis by revealing RNA processing intermediates. Compared to sequencing, however, northern blotting is a relatively insensitive technology. This creates a challenge for detecting low expressed miRNAs, particularly those produced by inefficient, non-canonical pathways. In this chapter, we describe a strategy to study such miRNAs by northern blotting that involves ectopic expression of both miRNAs and miRNA-binding Argonaute (Ago) proteins. Through use of epitope tags, this strategy also provides a convenient method for verification of small RNA competency to be loaded into regulatory complexes.

Detecting Agotrons in Ago CLIPseq Data.

Timely and accurate regulation of gene expression is essential to all organisms. MicroRNAs (miRNAs) are a well-characterized and important class of gene expression regulators. We recently identified a novel class of gene regulators, the agotrons. Agotrons derive from short introns and associate with Argonaute (Ago) proteins much similar to miRNAs. However, agotrons completely bypass the conventional miRNA biogenesis pathway and thus exist as full-length introns, which disobey the classical rules on Ago-substrate requirements. As a class, agotrons are conserved in mammals, and despite the non-canonical biogenesis pathway, agotrons maintain the ability to deregulate mRNAs with seed-matches in the 3'UTR. While several pipelines exist for the detection of miRNAs, no bioinformatics toolkit has yet been developed to specifically identify agotrons. Here, we describe a simple approach, termed agotron_detector ( https://github.com/ncrnalab/agotron_detector ), to identify and quantify agotrons in Ago CLIPseq datasets. Hopefully, this allows researchers worldwide to characterize agotrons in more detail and to reveal the biological relevance of these fascinating RNA species.

An Expanded Role for HLA Genes: HLA-B Encodes a microRNA that Regulates IgA and Other Immune Response Transcripts.

We describe a novel functional role for the HLA-B locus mediated by its intron-encoded microRNA (miRNA), miR-6891-5p. We show that in vitro inhibition of miR-6891-5p impacts the expression of nearly 200 transcripts within the B-lymphoblastoid cell line (B-LCL) COX, affecting a large number of metabolic pathways, including various immune response networks. The top affected transcripts following miR-6891-5p inhibition are those encoding the heavy chain of IgA. We identified a conserved miR-6891-5p target site on the 3'UTR of both immunoglobulin heavy chain alpha 1 and 2 (IGHA1 and IGHA2) transcripts and demonstrated that this miRNA modulates the expression of IGHA1 and IGHA2. B-LCLs from IgA-deficient patients expressed significantly elevated levels of miR-6891-5p when compared with unaffected family members. Upon inhibition of miR-6891-5p, IgA mRNA expression levels were increased, and IgA secretion was restored in the B-LCL of an IgA-deficient patient. These findings indicate that miR-6891-5p regulates IGHA1 and IGHA2 gene expression at the posttranscriptional level and suggest that increase in miR-6891-5p levels may contribute to the etiology of selective IgA deficiency.

Splicing-dependent expression of microRNAs of mirtron origin in human digestive and excretory system cancer cells.

BACKGROUND: An abundant class of intronic microRNAs (miRNAs) undergoes atypical Drosha-independent biogenesis in which the spliceosome governs the excision of hairpin miRNA precursors, called mirtrons. Although nearly 500 splicing-dependent miRNA candidates have been recently predicted via bioinformatic analysis of human RNA-Seq datasets, only a few of them have been experimentally validated. The detailed mechanism of miRNA processing by the splicing machinery and the roles of mirtronic miRNAs in cancer are yet to be uncovered. METHODS: We experimentally examined whether biogenesis of certain miRNAs is under a splicing control by analyzing their expression levels in response to alterations in the 5'- and 3'-splice sites of a series of intron-containing minigenes carrying appropriate miRNAs. The expression levels of the miRNAs processed from mirtrons were determined by quantitative real-time PCR in five digestive tract (pancreas PANC-1, SU.86.86, T3M4, stomach KATOIII, colon HCT116) and two excretory system (kidney CaKi-1, 786-O) carcinoma cell lines as well as in pancreatic, stomach, and colorectal tumors. Transiently expressed SRSF1 and SRSF2 splicing factors were quantified by western blotting in the nuclear fractions of HCT116 cells. RESULTS: We found that biogenesis of the human hsa-miR-1227-3p, hsa-miR-1229-3p, and hsa-miR-1236-3p is splicing-dependent; therefore, these miRNAs can be assigned to the class of miRNAs processed by a non-canonical mirtron pathway. The expression analysis revealed a differential regulation of human mirtronic miRNAs in various cancer cell lines and tumors. In particular, hsa-miR-1229-3p is selectively upregulated in the pancreatic and stomach cancer cell lines derived from metastatic sites. Compared with the healthy controls, the expression of hsa-miR-1226-3p was significantly higher in stomach tumors but extensively downregulated in colorectal tumors. Furthermore, we provided evidence that overexpression of SRSF1 or SRSF2 can upregulate the processing of individual mirtronic miRNAs in HCT116 cells. CONCLUSIONS: An interplay of different splicing factors, such as SRSF1 or SRSF2, may alter the levels of miRNAs of mirtron origin in a cell. Our findings underline the specific expression profiles of mirtronic miRNAs in colorectal, stomach, and pancreatic cancer.

Deep sequencing analyses of pine wood nematode Bursaphelenchus xylophilus microRNAs reveal distinct miRNA expression patterns during the pathological process of pine wilt disease.

Bursaphelenchus xylophilus is known as the causative agent of pine wilt disease with complex life cycles. In this research, four small RNA libraries derived from different infection stages of pine wilt disease were constructed and sequenced. Consequently, we obtained hundreds of evolutionarily conserved miRNAs and novel miRNA candidates. The analysis of miRNA expression patterns showed that most miRNAs were expressed at extraordinarily high levels during the middle stage of pine wilt disease. Functional analysis revealed that expression levels of miR-73 and miR-239 were mutually exclusive with their target GH45 cellulase genes. In addition, another set of atypical miRNAs, termed mirtrons, was also identified in this study. Thus, our research has provided detailed characterization of B. xylophilus miRNA expression patterns during the pathological process of pine wilt disease. These findings would contribute to more in-depth understanding of this devastating plant disease.

"Pocket-sized RNA-Seq": A Method to Capture New Mature microRNA Produced from a Genomic Region of Interest.

Currently, the discovery of new small ncRNAs requires high throughput methods even in the case of focused research on the regulation of specific genes or set of genes. We propose herein a simple, rapid, efficient, and cost effective method to clone and sequence single, yet unknown, small ncRNA. This technique that we called "Pocket-sized RNA-Seq" or psRNA-seq is based on in vitro transcription, RNA pull down and adapted RACE-PCR methods that allow its implementation using either available commercial kits or in-house reagents.

Expanding the annotation of zebrafish microRNAs based on small RNA sequencing.

MicroRNAs (miRs) are short non-coding RNAs that fine-tune the regulation of gene expression to coordinate a wide range of biological processes. Because of their role in the regulation of gene expression, miRs are essential players in development by acting on cell fate determination and progression towards cell differentiation and are increasingly relevant to human health and disease. Although the zebrafish Danio rerio is a major model for studies of development, genetics, physiology, evolution, and human biology, the annotation of zebrafish miR-producing genes remains limited. In the present work, we report deep sequencing data of zebrafish small RNAs from brain, heart, testis, and ovary. Results provide evidence for the expression of 56 un-annotated mir genes and 248 un-annotated mature strands, increasing the number of zebrafish mir genes over those already deposited in miRBase by 16% and the number of mature sequences by 63%. We also describe the existence of three pairs of mirror-mir genes and two mirtron genes, genetic features previously undescribed in non-mammalian vertebrates. This report provides information that substantially increases our knowledge of the zebrafish miRNome and will benefit the entire miR community.

The fascinating world of RNA interference.

Micro- and short-interfering RNAs represent small RNA family that are recognized as critical regulatory species across the eukaryotes. Recent high-throughput sequencing have revealed two more hidden players of the cellular small RNA pool. Reported in mammals and Caenorhabditis elegans respectively, these new small RNAs are named piwi-interacting RNAs (piRNAs) and 21U-RNAs. Moreover, small RNAs including miRNAs have been identified in unicellular alga Chlamydomonas reinhardtii, redefining the earlier concept of multi-cellularity restricted presence of these molecules. The discovery of these species of small RNAs has allowed us to understand better the usage of genome and the number of genes present but also have complicated the situation in terms of biochemical attributes and functional genesis of these molecules. Nonetheless, these new pools of knowledge have opened up avenues for unraveling the finer details of the small RNA mediated pathways.