Project description:Noncoding variants play a central role in the genetics of complex traits, but we still lack a full description of the main molecular pathways through which they act. Here we used molecular data to quantify the contribution of cis-acting genetic effects at each major stage of gene regulation from chromatin to proteins, within a population sample of Yoruba lymphoblastoid cell lines (LCLs). We performed 4sU metabolic labeled transcripts in 65 YRI LCLs to identify genetic variants that affect transcription rates. As expected, we found an important contribution of genetic variation via chromatin, contributing â¼65% of eQTLs (expression Quantitative Trait Loci). The remaining eQTLs, which are not asso- ciated with chromatin-level variation, are highly enriched in transcribed regions, and hence may affect expression through co- or post-transcriptional processes. International HapMap lymphoblastoid cell lines (LCLs) derived from YRI (Yoruba in Ibadan, Nigeria); We adapted the 4sU labelling method from (PMID 21516085). Briefly, cell cultures were grown to log phase in volumes sufficient to yield about 300 ng of 4sU-labeled RNA. Cells were incubated with 4sU for the required length of time (0, 30, or 60 minutes), then washed, pelleted, and frozen. Total RNA was extracted, and 4sU-labeled RNA was separated from total RNA using a bead-based biotin-streptavidin purification protocol. We sequenced metabolic labeled transcripts in 65 YRI LCLs 30 minutes and 60 minutes after incubation.
Project description:Noncoding variants play a central role in the genetics of complex traits, but we still lack a full description of the main molecular pathways through which they act. Here we used molecular data to quantify the contribution of cis-acting genetic effects at each major stage of gene regulation from chromatin to proteins, within a population sample of Yoruba lymphoblastoid cell lines (LCLs). We performed 4sU metabolic labeled transcripts in 65 YRI LCLs to identify genetic variants that affect transcription rates. As expected, we found an important contribution of genetic variation via chromatin, contributing ∼65% of eQTLs (expression Quantitative Trait Loci). The remaining eQTLs, which are not asso- ciated with chromatin-level variation, are highly enriched in transcribed regions, and hence may affect expression through co- or post-transcriptional processes.
Project description:We carried out an extensive functional genomics analysis to elucidate the roles, if any, of splicing at enhancer-associated lincRNA loci (elincRNA). While we found little evidence supporting conservation of elincRNA exons, their splice sites and other splicing associated motifs accumulated significantly fewer mutations than neutrally evolving regions, suggesting that selection has acted to preserve their splicing. We showed that in contrast to other lincRNAs, not only were multi-exonic elincRNAs efficiently and rapidly spliced, they were also specifically associated with enhanced expression of proximal genes and increased local chromosomal interactions. Enrichment of chromatin signatures associated with high enhancer activity at these loci further supports the contribution of elincRNA splicing to enhancer function. Our results elucidate the previously observed evolutionary constraint at elincRNA splice sites, but not their exons, and corroborate the impact of elincRNA splicing modulation on neighboring gene expression regulation, highlighting an unexpected contribution of elincRNA splicing to enhancer function.
Project description:To investigate the effect of increasing 4sU labeling concentrations on quantification bias in nucleotide conversion RNA-seq, we labeled 3 different cell lines with increasing concentrations of 4sU. We then measured 4sU dropout in 4sU samples compared to 4sU naive samples.
Project description:To investigate the effect of labeling durations on quantification bias in nucleotide conversion RNA-seq, we labeled NIH cells with 4sU. We then measured 4sU dropout in 4sU samples compared to 4sU naive samples.
Project description:To reveal fast-acting transcriptional effects of SWI/SNF inhibition in neuroblastoma cells, we performed 4sU metabolic labeling followed by RNA-seq in IMR-32, KELLY, and SK-N-DZ cells and compared the effects of BRM014 with DMSO vehicle control. We also compared these effects to auxin-induced degradation of SMARCA4 in engineered cells with the minimal auxin-induced degron (SMARCA4-mAID). Analysis of transcripts reveal that SMARCA4 inhibition leads to suppression of transcripts involved in the G1 checkpoint.
Project description:Measurements of metabolically labeled transcripts in single cells for estimation of mRNA synthesis and degradation rates throughout the cell cycle and intestinal organoid differentiation.
Project description:RNA synthesis and decay rates determine the steady-state levels of cellular RNAs. Metabolic tagging of newly transcribed RNA by 4-thiouridine (4sU) can reveal the relative contributions of RNA synthesis and decay rates. The kinetics of RNA processing, however, so far remained unresolved. Here, we show that ultra-short 4sU-tagging not only provides snap-shot pictures of eukaryotic gene expression but, when combined with progressive 4sU-tagging and RNA-seq, reveals global RNA processing kinetics at nucleotide resolution. Using this method, we identified classes of rapidly and slowly spliced/degraded introns. Interestingly, each class of splicing kinetics was characterized by a distinct association with intron length, gene length and splice site strength. For a large group of introns, we also observed long lasting retention in the primary transcript, but efficient secondary splicing or degradation at later time points. Finally, we show that processing of most, but not all small nucleolar (sno)RNA-containing introns is remarkably inefficient with the majority of introns being spliced and degraded rather than processed into mature snoRNAs. In summary, our study yields unparalleled insights into the kinetics of RNA processing and provides the tools to study molecular mechanisms of RNA processing and their contribution to the regulation of gene expression. 4sU-tagging was performed in human DG75 B-cells by adding 500 uM 4sU to cell culture medium for 5, 10, 15, 20 or 60 min. Following isolation of total cellular RNA, this was separated into nascent and untagged, pre-existing RNA. Nascent RNA as well as total and untagged RNA from 60 min 4sU-tagging were subjected to SOLiD sequencing (SOLiD II) obtaining 35 nt reads.
Project description:We identified the first Spt5-Pol II inhibitors (SPIs). SPIs faithfully reproduced Spt5 knockdown effects on proximal-promoter-pausing, NF-κB activation and the expanded-repeat huntingtin gene in neuronal cells. Using SPIs we identified Spt5 target genes that responded with profoundly diverse kinetics and a novel regulatory element of proximal-promoter-pausing. To validate that the effects of SPIs are at the transcriptional level, cellular RNA was metabolically labeled with 4-thio-uridine (4sU) for 2 hours in the presence of DMSO or SPI-21 in the presence or absence of TNFα. Newly synthesized labeled RNA was then purified and subjected to RNA-seq.