Dataset Information

Massively parallel functional annotation of 3' untranslated regions.

ABSTRACT: Functional characterization of noncoding sequences is crucial for understanding the human genome and learning how genetic variation contributes to disease. 3' untranslated regions (UTRs) are an important class of noncoding sequences, but their functions remain largely uncharacterized. We developed a method for massively parallel functional annotation of sequences from 3' UTRs (fast-UTR) and used this approach to measure the effects of a total of >450 kilobases of 3' UTR sequences from >2,000 human genes on steady-state mRNA abundance, mRNA stability and protein production. We found widespread regulatory effects on mRNA that were coupled to effects on mRNA stability and protein production. Furthermore, we discovered 87 novel cis-regulatory elements and measured the effects of genetic variation within known and novel 3' UTR motifs. This work shows how massively parallel approaches can improve the functional annotation of noncoding sequences, advance our understanding of cis-regulatory mechanisms and quantify the effects of human genetic variation.

SUBMITTER: Zhao W

PROVIDER: S-EPMC3981918 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Massively parallel functional annotation of 3' untranslated regions.

Zhao Wenxue W Pollack Joshua L JL Blagev Denitza P DP Zaitlen Noah N McManus Michael T MT Erle David J DJ

Nature biotechnology 20140316 4

Functional characterization of noncoding sequences is crucial for understanding the human genome and learning how genetic variation contributes to disease. 3' untranslated regions (UTRs) are an important class of noncoding sequences, but their functions remain largely uncharacterized. We developed a method for massively parallel functional annotation of sequences from 3' UTRs (fast-UTR) and used this approach to measure the effects of a total of >450 kilobases of 3' UTR sequences from >2,000 hum ...[more]

PMID: 24633241

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Project description:The adaptive immune system generates a remarkable range of antigen-specific T-cell receptors (TCRs), allowing the recognition of a diverse set of antigens. Most of this diversity is encoded in the complementarity determining region 3 (CDR3) of the ? chain of the ?? TCR, which is generated by somatic recombination of noncontiguous variable (V), diversity (D), and joining (J) gene segments. Deletion and non-templated insertion of nucleotides at the D-J and V-DJ junctions further increases diversity. Many of these gene segments are annotated as non-functional owing to defects in their primary sequence, the absence of motifs necessary for rearrangement, or chromosomal locations outside the TCR locus.We sought to utilize a novel method, based on high-throughput sequencing of rearranged TCR genes in a large cohort of individuals, to evaluate the use of functional and non-functional alleles. We amplified and sequenced genomic DNA from the peripheral blood of 587 healthy volunteers using a multiplexed polymerase chain reaction assay that targets the variable region of the rearranged TCR? locus, and we determined the presence and the proportion of productive rearrangements for each TCR? V gene segment in each individual. We then used this information to annotate the functional status of TCR? V gene segments in this cohort.For most TCR? V gene segments, our method agrees with previously reported functional annotations. However, we identified novel non-functional alleles for several gene segments, some of which were used exclusively in our cohort to the detriment of reported functional alleles. We also saw that some gene segments reported to have both functional and non-functional alleles consistently behaved in our cohort as either functional or non-functional, suggesting that some reported alleles were not present in the population studied.In this proof-of-principle study, we used high-throughput sequencing of the TCR? locus of a large cohort of healthy volunteers to evaluate the use of functional and non-functional alleles of individual TCR? V gene segments. With some modifications, our method has the potential to be extended to gene segments in the ?, ?, and ? TCR loci, as well as the genes encoding for B-cell receptor chains.

Dataset Information

Massively parallel functional annotation of 3' untranslated regions.

Publications

Massively parallel functional annotation of 3' untranslated regions.

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