The Integrator complex regulates microRNA abundance through RISC loading

MicroRNA (miRNA) homeostasis is crucial for the post-transcriptional regulation of their target genes and miRNA dysregulation has been linked to multiple diseases, including cancer. The molecular mechanisms underlying miRNA biogenesis from processing of primary miRNA transcripts to formation of mature miRNA duplex are well understood. Loading of miRNA duplex into members of the Argonaute (Ago) protein family, representing the core of the RNA-induced silencing complex (RISC), is pivotal to miRNA-mediated gene silencing. The Integrator complex has been previously shown to be an important regulator of RNA maturation, RNA polymerase II pause-release, and premature transcriptional termination. Here, we report that loss of Integrator results in global diminution of mature miRNAs. By incorporating 4-Thiouridine (s4U) in nascent transcripts, we traced miRNA fate from biogenesis to stabilization and identified Integrator to be essential for proper miRNA assembly into RISC. Enhanced UV crosslinking and immunoprecipitation (eCLIP) of Integrator confirms a robust association with mature miRNAs. Indeed, Integrator potentiates Ago2-mediated cleavage of target RNAs. These findings highlight an essential role for Integrator in miRNA abundance and RISC function.

Biogenesis of a 22-nt microRNA in Phaseoleae species by precursor-programmed uridylation

Phased, secondary siRNAs (phasiRNAs) represent a class of small RNAs in plants generated via distinct biogenesis pathways, predominantly dependent on the activity of 22-nt miRNAs. Most 22-nt miRNAs are processed by DCL1 from miRNA precursors containing an asymmetric bulge, yielding a 22/21-nt miRNA/miRNA* duplex. Here we show that miR1510, a soybean miRNA capable of triggering phasiRNA production from numerous nucleotide-binding leucine-rich repeat (NB-LRRs), previously described as 21 nt in its mature form, primarily accumulates as a 22-nt isoform via monouridylation. We demonstrate that, in Arabidopsis, this uridylation is performed by HESO1. Biochemical experiments showed that the 3′ terminus of miR1510 is only partially 2′-O-methylated because of the terminal mispairing in the miR1510/miR1510* duplex that inhibits HEN1 activity in soybean. miR1510 emerged in the Phaseoleae ∼41–42 million years ago with a conserved precursor structure yielding a 22-nt monouridylated form, yet a variant in mung bean is processed directly in a 22-nt mature form. This analysis of miR1510 yields two observations: (i) plants can utilize postprocessing modification to generate abundant 22-nt miRNA isoforms to more efficiently regulate target mRNA abundances; and (ii) comparative analysis demonstrates an example of selective optimization of precursor processing of a young plant miRNA.

Biogenesis of a young, 22-nt microRNA in Phaseoleae species by precursor-programmed uridylation

ABSTRACTPhased, secondary siRNAs (phasiRNAs) represent a class of small RNAs in plants generated via distinct biogenesis pathways, predominantly dependent on the activity of 22 nt miRNAs. Most 22 nt miRNAs are processed by DCL1 from miRNA precursors containing an asymmetric bulge, yielding a 22/21 nt miRNA/miRNA* duplex. Here we show that miR1510, a soybean miRNA capable of triggering phasiRNA production from numerous NB-LRRs, previously described as 21 nt in its mature form, primarily accumulates as a 22 nt isoform via monouridylation. We demonstrate that in Arabidopsis, this uridylation is performed by HESO1. Biochemical experiments showed that the 3’ terminus of miR1510 is only partially 2’-O-methylated, because of the terminal mispairing in the miR1510/miR1510* duplex that inhibits HEN1 activity in soybean. miR1510 emerged in the Phaseoleae ~41 to 42 MYA with a conserved precursor structure yielding a 22 nt monouridylated form, yet a variant in mung bean is processed directly in a 22 nt mature form. This analysis of miR1510 yields two observations: (1) plants can utilize post-processing modification to generate abundant 22 nt miRNA isoforms to more efficiently regulate target mRNA abundances; (2) comparative analysis demonstrates an example of selective optimization of precursor processing of a young plant miRNA.

Differential Expression of Mirna* Species in Cancer and the Contribution of MiR-223* to the Development of Acute Myeloid Leukemia.

Abstract 2960 Poster Board II-936 Processing of the pre-miRNA through Dicer1 generates a miRNA duplex, consisting of a miRNA and miRNA* strand. While the functional roles of miRNAs are now well established, the potential roles of miRNA* species remain unclear. However, recent evidence suggests that the star strand of some miRNAs can be abundant and enter the RISC complex. Since the abundance of miRNA*s has not been comprehensively assessed in mammals and we took advantage of 10 deep sequencing libraries from a variety of human and murine cells to determine the most abundant complementary strand for non-annotated miRNA*s. We then calculated the ratio of miRNA/miRNA* for each miRNA duplex. In contrast to previous assumptions that one strand is highly dominant, we found that approximately 50% of the investigated miRNA duplexes exhibit high ratios with a dominating strand (ratio >100), 20% have intermediate ratios (ratio between 100-10) and a remarkable 10% show low ratios (ratio <10), indicating comparable expression of both strands. In addition, we found that ∼10% of all miRNA/miRNA* duplexes display inverse ratios (ratio<1), indicating incorrect annotation in miRBase. Comparing miRNA/miRNA* ratios across the miRNA sequence libraries revealed that most ratios remain constant across tissues and species. This could possibly allow for a novel classification of miRNAs into a-duplexes, miRNAs duplexes with a dominant strand and b-duplexes with both strands being abundant. However, certain ratios were highly variable across the libraries examined as exemplified for the ratio of miR-223/miR-223* which ranged from 0.11 (317:2684 read counts) to 19.6 (13006:660 read counts) in murine and human leukemia cell lines. Bioinformatics analysis on predicted miR-223* targets showed an enrichment for cancer associated genes (p<0.05), suggesting a tumor suppressor-like role for miR-223. Consistent with this, an analysis of samples from 94 AML patients with normal karyotype revealed an inverse correlation of miR-223* with CD34 expression (p=0.018), a negative prognostic marker in AML. In addition, in vitro experiments with mutated miR-223 and miR-223* constructs revealed regulatory potential for miR-223* in myeloid progenitor cells. Taken together, we propose a new classification for miRNA duplexes and provide evidence for a possible role a miRNA* in the development of acute myeloid leukemia. Disclosures: No relevant conflicts of interest to declare.