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Curr Protoc Microbiol. 2016 May 6;41:14E.7.1-14E.7.18. doi: 10.1002/cpmc.1.

PA-seq for Global Identification of RNA Polyadenylation Sites of Kaposi's Sarcoma-Associated Herpesvirus Transcripts.

Author information

1
Ministry of Education (MOE) Key Laboratory of Contemporary Anthropology and State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, School of Life Sciences, Fudan University, Shanghai, People's Republic of China.
2
These authors should be considered co-first authors.
3
Tumor Virus RNA Biology Section, Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland.
4
Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland.
5
Corresponding author.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncovirus linked to the development of several malignancies in immunocompromised patients. Like other herpesviruses, KSHV has a large DNA genome encoding more than 100 distinct gene products. Despite being transcribed and processed by cellular machinery, the structure and organization of KSHV genes in the virus genome differ from what is observed in cellular genes from the human genome. A typical feature of KSHV expression is the production of polycistronic transcripts initiated from different promoters but sharing the same polyadenylation site (pA site). This represents a challenge in determination of the 3' end of individual viral transcripts. Such information is critical for generation of a virus transcriptional map for genetic studies. Here we present PA-seq, a high-throughput method for genome-wide analysis of pA sites of KSHV transcripts in B lymphocytes with latent or lytic KSHV infection. Besides identification of all viral pA sites, PA-seq also provides quantitative information about the levels of viral transcripts associated with each pA site, making it possible to determine the relative expression levels of viral genes at various stages of infection. Due to the indiscriminate nature of PA-seq, the pA sites of host transcripts are also concurrently mapped in the testing samples. Therefore, this technology can simultaneously estimate the expression changes of host genes and RNA polyadenylation upon KSHV infection.

KEYWORDS:

KSHV; PA-seq; herpesvirus; polyadenylation; transcript

PMID:
27153384
PMCID:
PMC4910627
DOI:
10.1002/cpmc.1
[Indexed for MEDLINE]
Free PMC Article

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