Functional Atlas of Primary miRNA Maturation by the Microprocessor

Rice, Greggory M.; Shivashankar, Varun; Ma, Eric; Baryza, Jeremy; Nutiu, Razvan

doi:10.1016/j.molcel.2020.10.028

Cited by 32 publications

(45 citation statements)

References 48 publications

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“…Pri-miRNAs are long double-stranded RNAs composed of an apical loop, a stem of ~35 base pairs, and two flanking single-stranded nucleotides (Belt and Wedge) at the basal end [25]. Then, pri-miRNAs are further cleaved into pre-miRNAs by a microprocessor complex containing the RNase III enzyme DROSHA and RNA binding protein DiGeorge syndrome critical region 8 (DGCR8) in the nucleus [25,26]. During this cleavage event, the DGCR8 double-stranded RNA binding domain (dsRBD) in the apical half and the DROSHA dsRBD in the basal half form a "double-dsRBD" molecular ruler for an ~35 base pairs stem region [25].…”

Section: Introductionmentioning

confidence: 99%

Subcellular Localization of miRNAs and Implications in Cellular Homeostasis

Jie

Feng

Huang

et al. 2021

Genes

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MicroRNAs (miRNAs) are thought to act as post-transcriptional regulators in the cytoplasm by either dampening translation or stimulating degradation of target mRNAs. With the increasing resolution and scope of RNA mapping, recent studies have revealed novel insights into the subcellular localization of miRNAs. Based on miRNA subcellular localization, unconventional functions and mechanisms at the transcriptional and post-transcriptional levels have been identified. This minireview provides an overview of the subcellular localization of miRNAs and the mechanisms by which they regulate transcription and cellular homeostasis in mammals, with a particular focus on the roles of phase-separated biomolecular condensates.

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Section: Introductionmentioning

confidence: 99%

Subcellular Localization of miRNAs and Implications in Cellular Homeostasis

Jie

Feng

Huang

et al. 2021

Genes

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“…Intriguingly, the lower basal stem stability is ranked as the most important individual feature using MapToCleave data and the second most important using in vivo data (in green, Figure 7B), suggesting it is at least as important for processing as are the well-studied sequence motifs. We find that Shannon entropy (Rice et al, 2020) explains little of in vivo processing (in grey, Figure 7B), but does contribute to processing in our cleavage assay, although to a lesser extent than the lower basal stem stability (Figure 7B). Interestingly, two bulge-depleted regions of the precursors also contribute (in blue), consistent with previous results (Roden et al, 2017), as does the stability of other local structures along the miRNA stem that have only been investigated in a few studies (Li et al, 2020a; Nguyen et al, 2020).…”

Section: Resultsmentioning

confidence: 88%

“…A major advantage of MapToCleave is the ability to measure miRNA precursor processing in living cells, in the natural environment of protein cofactors, cellular compartments and more, in contrast to previous large-scale efforts to profile miRNA biogenesis which have all been in vitro (Auyeung et al, 2013; Fang and Bartel, 2015; Feng et al, 2011; Kwon et al, 2019; Li et al, 2020a; Nguyen et al, 2020; Rice et al, 2020). As a proof-of-principle, we tested human and murine MapToCleave precursors in human embryonic kidney HEK-293T cells and mouse NIH-3T3 fibroblast cells (Methods).…”

Section: Resultsmentioning

confidence: 99%

“…Previous studies have been limited in that variants of only a few miRNA precursors have been tested, leaving open the possibility that some important biogenesis features may remain undiscovered. One recent study partly overcame this limitation by testing thousands of distinct RNA structures at the same time, providing evidence that structural uncertainty, measured as Shannon entropy, negatively influences processing (Rice et al, 2020). However, this experiment was conducted in vitro , so the contribution of the cellular context to miRNA biogenesis remains unstudied on a large scale.…”

Section: Introductionmentioning

confidence: 99%

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MapToCleave: high-throughput profiling of microRNA biogenesis in living cells

Kang

Fromm

Houben

et al. 2021

Preprint

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Previous large-scale studies have uncovered many features that determine the processing of microRNA (miRNA) precursors, however, they have been conducted in vitro. Here we introduce MapToCleave, a new method to simultaneously profile processing of thousands of distinct RNA structures in living cells. Our new in cell method captures essentially all the biogenesis features that have been discovered through near two decades of in vitro studies - providing support for both approaches. We find that miRNA precursors with a stable lower basal stem are more efficiently processed and also have higher expression in vivo in tissues from twenty animal species. We systematically compare the importance of known and novel sequence and structural features and test biogenesis of miRNA precursors from ten animal and plant species in human cells. Lastly, we provide evidence that the GHG motif better predicts processing when defined as a structure rather than sequence motif, consistent with recent cryo-EM studies. In summary, we apply a new screening assay in living cells to reveal the importance of lower basal stem stability for miRNA processing and in vivo expression.

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“…We previously determined hairpin optimality features for each KSHV pre-miRNA ( 31), which we decided to update in order to take into account novel features that have been published since (46,47). In particular, we calculated the Shannon entropy for each miRNA hairpin as highlighted in Figure S6.…”

Section: Clustered Kshv Pre-mirnas Are Processed In Vitro By the Microprocessor With Different Efficienciesmentioning

confidence: 99%

Cisregulation within a cluster of viral microRNAs

Vilimova

Contrant

Randrianjafy

et al. 2020

Preprint

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MicroRNAs (miRNAs) are small regulatory RNAs involved in virtually all biological processes. Although a large number of them are co-expressed from clusters, little is known regarding the impact of this organization on the regulation of their accumulation. In this study, we set to decipher the regulatory mechanism controlling the expression of the ten clustered pre-miRNAs from Kaposi’s sarcoma associated herpesvirus (KSHV). We measured in vitro the efficiency of cleavage of each individual pre-miRNA by the Microprocessor. Strikingly, pre-miR-K1 and -K3 were the most efficiently cleaved pre-miRNAs but their mature forms accumulated at low levels, indicating that they might perform an additional function. A mutational analysis showed that they are indeed important for the optimal expression of the whole set of miRNAs. We showed that this solely relies on the presence of a canonical pre-miRNA at this localization since we could functionally replace pre-miR-K1 by a heterologous pre-miRNA. Further in vitro processing analysis suggests that the two stem-loops act in cis and that the cluster is cleaved in a sequential manner. Finally, we showed that we can exploit this feature of the cluster to inhibit the expression of the whole set of miRNAs by targeting the pre-miR-K1 with LNA-based antisense oligonucleotides.

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Functional Atlas of Primary miRNA Maturation by the Microprocessor

Cited by 32 publications

References 48 publications

Subcellular Localization of miRNAs and Implications in Cellular Homeostasis

Subcellular Localization of miRNAs and Implications in Cellular Homeostasis

MapToCleave: high-throughput profiling of microRNA biogenesis in living cells

Cisregulation within a cluster of viral microRNAs

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