Discovering RNA-Protein Interactome by Using Chemical Context Profiling of the RNA-Protein Interface

Parisien, Marc; Wang, Xiaoyun; Perdrizet, George A.; Lamphear, Corissa L.; Fierke, Carol A.; Maheshwari, Ketan; Wilde, Michael; Sosnick, Tobin R.; Pan, Tao

doi:10.1016/j.celrep.2013.04.010

Cited by 22 publications

(28 citation statements)

References 67 publications

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“…Using 3 ′ 32 P-labeled total tRNA from HEK293T cells, hydrazine/aniline treatment yielded three major fragments that are consistent with the cleavage products of tRNA Ser at m 3 C32, tRNA Thr at m 3 C32, and tRNA Ser at m 3 C47d, and the LeuCAG site at m 3 C47d (Supplemental Fig. S5E; Parisien et al 2013). These specific tRNA products were confirmed by tRNA microarrays (Supplemental Fig.…”

Section: Identification Of Trna Methylation Sitesmentioning

confidence: 93%

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tRNA base methylation identification and quantification via high-throughput sequencing

Clark¹,

Evans²,

Dominissini

et al. 2016

RNA

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Eukaryotic transfer RNAs contain on average 14 modifications. Investigations of their biological functions require the determination of the modification sites and the dynamic variations of the modification fraction. Base methylation represents a major class of tRNA modification. Although many approaches have been used to identify tRNA base methylations, including sequencing, they are generally qualitative and do not report the information on the modification fraction. Dynamic mRNA modifications have been shown to play important biological roles; yet, the extent of tRNA modification fractions has not been reported systemically. Here we take advantage of a recently developed high-throughput sequencing method (DM-tRNA-seq) to identify and quantify tRNA base methylations located at the Watson-Crick face in HEK293T cells at single base resolution. We apply information derived from both base mutations and positional stops from sequencing using a combination of demethylase treatment and cDNA synthesis by a thermophilic reverse transcriptase to compile a quantitative "Modification Index" (MI) for six base methylations in human tRNA and rRNA. MI combines the metrics for mutational and stop components from alignment of sequencing data without demethylase treatment, and the modifications are validated in the sequencing data upon demethylase treatment. We identify many new methylation sites in both human nuclear and mitochondrial-encoded tRNAs not present in the RNA modification databases. The potentially quantitative nature of the MI values obtained from sequencing is validated by primer extension of several tRNAs. Our approach should be widely applicable to identify tRNA methylation sites, analyze comparative fractional modifications, and evaluate the modification dynamics between different samples.

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Section: Identification Of Trna Methylation Sitesmentioning

confidence: 93%

“…Poly(A) RNA and salmon sperm DNA were added to aid in precipitation. The fragments were then hybridized to custom-made tRNA microarrays at 60°C for 16 h and exposed to phosphorimaging as previously described (Parisien et al 2013). …”

Section: Lc/ms-qqqmentioning

confidence: 99%

tRNA base methylation identification and quantification via high-throughput sequencing

Clark¹,

Evans²,

Dominissini

et al. 2016

RNA

Self Cite

167

224

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“…10,11 tRNAs have further been reported to serve as a source of small functional RNAs. [12][13][14] Numerous tRNA-interacting proteins have recently been proposed, 15 indicating the scope of tRNA biological functions may be far beyond that previously assumed.…”

Section: Introductionmentioning

confidence: 99%

Four-leaf clover qRT-PCR: A convenient method for selective quantification of mature tRNA

et al. 2015

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Transfer RNAs (tRNAs) play a central role in translation and also recently appear to have a variety of other functions in biological processes beyond translation. Here we report the development of Four-Leaf clover qRT-PCR (FL-PCR), a convenient PCR-based method, which can specifically quantify individual mature tRNA species. In FL-PCR, T4 RNA ligase 2 specifically ligates a stem-loop adapter to mature tRNAs but not to precursor tRNAs or tRNA fragments. Subsequent TaqMan qRT-PCR amplifies only unmodified regions of the tRNA-adapter ligation products; therefore, FL-PCR quantification is not influenced by tRNA post-transcriptional modifications. FL-PCR has broad applicability for the quantification of various tRNAs in different cell types, and thus provides a much-needed simple method for analyzing tRNA abundance and heterogeneity.

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“…To investigate further tRNA species that bind to Mili, we performed a modified cross-linked immunoprecipitation-microarray (Clip-chip) analysis (22). This analysis was preferred to transcriptome sequencing (RNA-seq), which would not reveal correctly relative levels of tRNAs of low abundance, i.e., rare tRNA species.…”

Section: Resultsmentioning

confidence: 99%

“…The tRNA species that interacted specifically with Hili were determined by Clip-chip experiments as previously described (22). 293T.mili cells were UV irradiated (400 mJ/cm 2 at 254 nm).…”

Section: Methodsmentioning

confidence: 99%

Hili Inhibits HIV Replication in Activated T Cells

Peterlin

Liu

Wang

et al. 2017

J Virol

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P-element-induced wimpy-like (Piwil) proteins restrict the replication of mobile genetic elements in the germ line. They are also expressed in many transformed cell lines. In this study, we discovered that the human Piwil 2 (Hili) protein can also inhibit HIV replication, especially in activated CD4+ T cells that are the preferred target cells for this virus in the infected host. Although resting cells did not express Hili, its expression was rapidly induced following T cell activation. In these cells and transformed cell lines, depletion of Hili increased levels of viral proteins and new viral particles. Further studies revealed that Hili binds to tRNA. Some of the tRNAs represent rare tRNA species, whose codons are overrepresented in the viral genome. Targeting tRNAArg(UCU) with an antisense oligonucleotide replicated effects of Hili and also inhibited HIV replication. Finally, Hili also inhibited the retrotransposition of the endogenous intracysternal A particle (IAP) by a similar mechanism. Thus, Hili joins a list of host proteins that inhibit the replication of HIV and other mobile genetic elements. IMPORTANCE Piwil proteins inhibit the movement of mobile genetic elements in the germ line. In their absence, sperm does not form and male mice are sterile. This inhibition is thought to occur via small Piwi-interacting RNAs (piRNAs). However, in some species and in human somatic cells, Piwil proteins bind primarily to tRNA. In this report, we demonstrate that human Piwil proteins, especially Hili, not only bind to select tRNA species, including rare tRNAs, but also inhibit HIV replication. Importantly, T cell activation induces the expression of Hili in CD4+ T cells. Since Hili also inhibited the movement of an endogenous retrovirus (IAP), our finding shed new light on this intracellular resistance to exogenous and endogenous retroviruses as well as other mobile genetic elements.

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Discovering RNA-Protein Interactome by Using Chemical Context Profiling of the RNA-Protein Interface

Cited by 22 publications

References 67 publications

tRNA base methylation identification and quantification via high-throughput sequencing

tRNA base methylation identification and quantification via high-throughput sequencing

Four-leaf clover qRT-PCR: A convenient method for selective quantification of mature tRNA

Hili Inhibits HIV Replication in Activated T Cells

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