Targeted RNA sequencing reveals the deep complexity of the human transcriptome

Mercer, Tim R.; Gerhardt, Daniel J.; Dinger, Marcel E.; Crawford, Joanna; Trapnell, Cole; Jeddeloh, Jeffrey A.; Mattick, John S.; Rinn, John L.

doi:10.1038/nbt.2024

Cited by 442 publications

(388 citation statements)

References 38 publications

Supporting

Mentioning

372

Contrasting

Unclassified

Order By: Relevance

“…Capture sequencing was performed similarly to previously described methods (Mercer et al 2012(Mercer et al , 2014 …”

Section: Capture Experimentsmentioning

confidence: 99%

“…We recently developed RNA Capture Sequencing (CaptureSeq), a technique that focuses sequencing on targeted RNA transcripts (Mercer et al 2012(Mercer et al , 2014. This approach is ideal for achieving high sequencing coverage of transient intronic species to resolve branchpoint sites.…”

Section: Intron Lariat Sequencing and Alignmentmentioning

confidence: 99%

“…We interrogated 238,576 introns (90.2% of annotated total) in this manner. We performed CaptureSeq similarly to previously described methods (Mercer et al 2012) with total RNA harvested from three biological replicates of human K562 cells (see Methods). We achieved greater than 100-fold enrichment of targeted RNA as determined by qPCR and analysis of RNA spike-in controls, corresponding to ;10.6-fold more reads (;133 million reads) that align to introns relative to matched RNAseq controls (Supplemental Fig.…”

Section: Intron Lariat Sequencing and Alignmentmentioning

confidence: 99%

See 2 more Smart Citations

Genome-wide discovery of human splicing branchpoints

et al. 2015

Self Cite

View full text Add to dashboard Cite

During the splicing reaction, the 59 intron end is joined to the branchpoint nucleotide, selecting the next exon to incorporate into the mature RNA and forming an intron lariat, which is excised. Despite a critical role in gene splicing, the locations and features of human splicing branchpoints are largely unknown. We use exoribonuclease digestion and targeted RNA-sequencing to enrich for sequences that traverse the lariat junction and, by split and inverted alignment, reveal the branchpoint. We identify 59,359 high-confidence human branchpoints in >10,000 genes, providing a first map of splicing branchpoints in the human genome. Branchpoints are predominantly adenosine, highly conserved, and closely distributed to the 39 splice site. Analysis of human branchpoints reveals numerous novel features, including distinct features of branchpoints for alternatively spliced exons and a family of conserved sequence motifs overlapping branchpoints we term B-boxes, which exhibit maximal nucleotide diversity while maintaining interactions with the keto-rich U2 snRNA. Different B-box motifs exhibit divergent usage in vertebrate lineages and associate with other splicing elements and distinct intron-exon architectures, suggesting integration within a broader regulatory splicing code. Lastly, although branchpoints are refractory to common mutational processes and genetic variation, mutations occurring at branchpoint nucleotides are enriched for disease associations.[Supplemental material is available for this article.]The majority of human genes are spliced, a process whereby introns are removed from the nascent RNA and the remaining exonic sequence joined together into a mature RNA transcript. In addition, alternative splicing generates complex networks of isoforms from human gene loci and plays a major role in shaping the diversity of the transcriptome (Kapranov et al. 2005;Gerstein et al. 2007;Djebali et al. 2012).Splicing occurs in the spliceosome, a large ribonucleoprotein complex that recognizes at least three genetic elements within each intron: the 59 splice site (59SS), the 39 splice site (39SS), and the branchpoint (Will and L€ uhrmann 2011). RNU2-1, the U2 spliceosomal RNA (snRNA) base pairs to the sequence surrounding the unpaired branchpoint nucleotide, which then undergoes transesterification with the 59 end of the intron to form a closed lariat structure. The spliceosome then scans for the downstream 39 splice site, which undergoes a second trans-esterification reaction to join together the two exon ends and excise the intron lariat (Fig.

show abstract

“…Capture sequencing was performed similarly to previously described methods (Mercer et al 2012(Mercer et al , 2014 …”

Section: Capture Experimentsmentioning

confidence: 99%

Section: Intron Lariat Sequencing and Alignmentmentioning

confidence: 99%

Section: Intron Lariat Sequencing and Alignmentmentioning

confidence: 99%

See 1 more Smart Citation

Genome-wide discovery of human splicing branchpoints

et al. 2015

Self Cite

View full text Add to dashboard Cite

show abstract

“…While lncRNAs have been shown to have a broad range of biological functions both in the cytoplasm and the nucleus, a large number of nuclear lncRNAs were shown to originate from distal regulator elements (enhancers) to positively regulate gene expression [7][8][9]. Interestingly, while lncRNAs display tissue-specific expression patterns [10], they have a low evolutionary conservation rate between species [11].…”

mentioning

confidence: 99%

Where long noncoding RNAs meet DNA methylation

Lai

Shiekhattar

2014

Cell Res

View full text Add to dashboard Cite

“…107 However, we argue here that omitting the analysis of unspliced macro lncRNAs leads to a gap in our knowledge about this potential new layer of cis-regulatory information in the mammalian genome. The remarkable tissue-specificity of lncRNAs 67 strongly argues that they represent a functional layer of the transcriptome, rather than transcriptional noise (see discussion in ref.…”

Section: Resultsmentioning

confidence: 86%