Microprocessor Activity Controls Differential miRNA Biogenesis In Vivo

Conrad, Thomas; Marsico, Annalisa; Gehre, Maja; Ørom, Ulf Andersson

doi:10.1016/j.celrep.2015.01.064

Cited by 14 publications

(31 citation statements)

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“…We calculate the splicing index value θ (Mukherjee et al, 2017) (Supplementary Figure 2A) to determine splicing efficiency across all time points for introns that have at least 5 reads coverage on both 5’ and 3’ SJ for all included RNA sequencing libraries (four time points of BrU-pulse-chase-seq and the three Input samples for TNT-seq), yielding 13,532 introns with an extracted θ value ranging from 0 (unspliced) to 1 (fully spliced) (Methods). As expected, the cumulative distribution of the splicing index at 0 min, representing nascent RNA, precedes that of the steady-state chromatin-associated RNA (Conrad et al, 2014) indicating that pre-mRNA is efficiently captured by the assay (Figure 2A). Using k-means clustering with k = 3 we called three clusters of distinct splicing efficiency dynamics (SED) representing 4,882 fast, 5,702 medium, and 2,948 slowly processed introns (Figure 2B and Supplementary Figure 2B-D) (for the definition of SED see Methods under ‘Splicing kinetics and predictive models’).…”

Section: Resultssupporting

confidence: 74%

“… (a) Cumulative distribution of the splicing efficiency index from chromatin-associated RNA seq(Conrad et al, 2014), BrU Chase Seq 0 min, BrU Chase Seq 15min, BrU Chase Seq 30min and BrU Chase Seq 60 min (b) Violin plot representing the density of the distribution of the splicing efficiency index (θ value) with embedded box and whisker plots for introns grouped on the basis of differential splicing kinetics (see also Supplementary Figure 2A-D). (c-d) Average m6A signal per nucleotide position in the window +/- 500 nt around ( c ) 5’SJ and 3’SJ ( d ) for 13,532 introns.…”

Section: Resultsmentioning

confidence: 99%

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Transient N-6-methyladensosine Transcriptome sequencing reveals a regulatory role of m6A in splicing efficiency

Louloupi

Ntini

Conrad

et al. 2018

Preprint

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Splicing efficiency varies among transcripts, and tight control of splicing kinetics is crucial for coordinated gene expression. N-6-methyladenosine (m6A) is the most abundant RNA modification and is involved in regulation of RNA biogenesis and function. The impact of m6A on the regulation of RNA splicing kinetics has not been investigated. Here, we provide the first time-resolved high-resolution assessment of m6A on nascent RNA transcripts and unveil its importance for the control of RNA splicing kinetics. We identify that early cotranscriptional m6A deposition near splice junctions promotes fast splicing, while m6A modification of introns is associated with long, slowly processed introns and alternative splicing events. In conclusion, by directly comparing the processing dynamics of individual transcripts in the methylated versus unmethylated state on a transcriptome-wide scale we show that early m6A deposition marks transcripts for a fast-track processing.

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Section: Resultssupporting

confidence: 74%

Section: Resultsmentioning

confidence: 99%

Transient N-6-methyladensosine Transcriptome sequencing reveals a regulatory role of m6A in splicing efficiency

Louloupi

Ntini

Conrad

et al. 2018

Preprint

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“…ssHMM also reflects a strong preference for the stem context which is in accordance with the structural motif found by GraphProt and the fact that DGCR8 contains two doublestranded RNA-binding domains [46,44]. This is in line with the findings from two previous studies [47,48] aimed at characterizing sequence-structure determinants of efficient pri-miRNA processing. These studies identified a basal, highly-conserved UG dinucleotide motif in a stem context at RNA positions -14 and -13 from the Drosha cleavage sites to be involved in enhanced pri-miRNA processing.…”

Section: Dgcr8supporting

confidence: 87%

ssHMM: Extracting intuitive sequence-structure motifs from high-throughput RNA-binding protein data

Heller

Krestel

Ohler

et al. 2016

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RNA-binding proteins (RBPs) play an important role in RNA post-transcriptional regulation and recognize target RNAs via sequence-structure motifs. The extent to which RNA structure influences protein binding in the presence or absence of a sequence motif is still poorly understood. Existing RNA motif finders either take the structure of the RNA only partially into account, or employ models which are not directly interpretable as sequence-structure motifs. We developed ssHMM, an RNA motif finder based on a hidden Markov model (HMM) and Gibbs sampling which fully captures the relationship between RNA sequence and secondary structure preference of a given RBP. Compared to previous methods which output separate logos for sequence and structure, it directly produces a combined sequence-structure motif when trained on a large set of sequences. ssHMM's model is visualized intuitively as a graph and facilitates biological interpretation. ssHMM can be used to find novel bona fide sequence-structure motifs of uncharacterized RBPs, such as the one presented here for the YY1 protein. ssHMM reaches a high motif recovery rate on synthetic data, it recovers known RBP motifs from CLIP-Seq data, and scales linearly on the input size, being considerably faster than MEMERIS and RNAcontext on large datasets while being on par with GraphProt. It is freely available on Github and as a Docker image.

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“…Analysis of chromatin-association of different groups of long ncRNAs based on epigenetic marks suggests that chromatin-release is involved in the mechanism of function of long ncRNAs engaged in strong chromatin interactions (Figure 2A-B). To address whether the regulatory effect of the long ncRNA happens while associated to chromatin or at or after dissociation from the chromatin-associated site of transcription, we fractionated MCF-7 cells, treated with either control or A-ROD siRNA, as described in Supplementary Methods (Conrad et al, 2014) (Supplementary Figure S5), and analyzed the nucleoplasmic and chromatin-associated RNA. While the chromatin-associated fraction is poorly targeted by siRNAs (Figure 5A), the nucleoplasmic A-ROD can be readily targeted by siRNA (Figure 5B), in agreement with recent studies showing assembly and function of the siRNA machinery in the nucleus (Gagnon et al, 2014).…”

Section: Resultsmentioning

confidence: 99%

Chromatin-release of the long ncRNA A-ROD is required for transcriptional activation of its target gene DKK1

Ntini

et al. 2017

Preprint

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Long non-coding RNAs (ncRNAs) are involved in both positive and negative regulation of transcription. Long ncRNAs are often enriched in the nucleus and at chromatin but whether chromatin-release plays a functional role is unknown. Here, we used epigenetic marks, expression level and strength of chromatin interactions to group long ncRNAs and find that those engaged in strong chromatin interactions are less enriched at chromatin in MCF-7 cells, suggesting a functional involvement of chromatin-release of long ncRNAs in transcriptional regulation. To study this further, we identify the long ncRNA A-ROD, an activating regulator of the Wnt signaling inhibitor DKK1. We show that A-ROD enhances transcription elongation of DKK1 in an RNA-dependent manner and that A-ROD recruits EBP1 to the DKK1 promoter. Our data suggest that the activating function depends on the release of A-ROD from chromatin, and further identify a functional regulatory interaction mediated by A-ROD in the transcription activation of DKK1. We propose that the release of a subset of long ncRNAs is important for their function, adding a new mechanistic perspective to the subcellular localization of long ncRNAs.

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Microprocessor Activity Controls Differential miRNA Biogenesis In Vivo

Cited by 14 publications

References 0 publications

Transient N-6-methyladensosine Transcriptome sequencing reveals a regulatory role of m6A in splicing efficiency

Transient N-6-methyladensosine Transcriptome sequencing reveals a regulatory role of m6A in splicing efficiency

ssHMM: Extracting intuitive sequence-structure motifs from high-throughput RNA-binding protein data

Chromatin-release of the long ncRNA A-ROD is required for transcriptional activation of its target gene DKK1

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