Mod5 protein binds to tRNA gene complexes and affects local transcriptional silencing

Pratt-Hyatt, Matthew; Pai, Dave A.; Haeusler, Rebecca A.; Wozniak, Glenn G.; Good, Paul D.; Miller, Erin; McLeod, Ian X.; Yates, John R.; Hopper, Anita K.; Engelke, David R.

doi:10.1073/pnas.1219946110

Cited by 31 publications

(46 citation statements)

References 56 publications

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“…Some active tRNA genes have been shown to possess chromatin insulator activity (35,81,82). In addition, extensive work in yeast has demonstrated that active tRNA genes can repress neighboring Pol II genes through tgm silencing (34,83,84). To understand whether AGO2 plays a role in either of these processes, we focused on a region on the p arm of human chromosome 17 that has been previously characterized to have active tRNA genes that can act as insulators (35).…”

Section: Human Argonaute 2 Binds To Actively Transcribed Trna Genesmentioning

confidence: 99%

“…Interestingly, the RNAi machinery has also been linked to chromatin insulator function in D. melanogaster, where it has been shown that AGO2 binds to insulator proteins and is critical for Fab-8 insulator function (4). In addition to roles in chromatin insulation, studies in yeast have shown that actively transcribed tRNA genes can repress neighboring Pol II gene transcription in a mechanism termed tRNA genemediated (tgm) silencing (32)(33)(34). Despite extensive work in yeast, it remains unknown whether tgm silencing is conserved in metazoans.…”

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confidence: 99%

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Argonaute 2 Binds Directly to tRNA Genes and Promotes Gene Repression in cis

Woolnough

Atwood

Giles

2015

Molecular and Cellular Biology

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To further our understanding of the RNAi machinery within the human nucleus, we analyzed the chromatin and RNA binding of Argonaute 2 (AGO2) within human cancer cell lines. Our data indicated that AGO2 binds directly to nascent tRNA and 5S rRNA, and to the genomic loci from which these RNAs are transcribed, in a small RNA-and DICER-independent manner. AGO2 chromatin binding was not observed at non-TFIIIC-dependent RNA polymerase III (Pol III) genes or at extra-TFIIIC (ETC) sites, indicating that the interaction is specific for TFIIIC-dependent Pol III genes. A genome-wide analysis indicated that loss of AGO2 caused a global increase in mRNA expression level among genes that flank AGO2-bound tRNA genes. This effect was shown to be distinct from that of the disruption of DICER, DROSHA, or CTCF. We propose that AGO2 binding to tRNA genes has a novel and important regulatory role in human cells. The RNA interference (RNAi) machinery has several important functions that are conserved from fission yeast to humans. In the cytoplasm of eukaryotic cells, Argonaute proteins promote degradation and/or translational repression of specific mRNAs through tethering by a complementary small RNA (1). The nuclear functions of the RNAi machinery appear to be diverse across species and include roles in promoting heterochromatin formation, regulating alternative splicing, and promoting insulator function (2-5). The ability of both Argonaute 1 (AGO1) and AGO2 to alter splicing in human cells depends upon small RNAs derived from exonic sequence, which tether Argonaute proteins to nascent transcripts (3, 6). Tethering of each Argonaute protein was shown to be capable of recruiting a histone lysine methyltransferase, which altered the RNA polymerase II (Pol II) elongation rate and thus facilitated alternative splicing. This interaction between Argonaute proteins and the transcriptional apparatus is conserved in Drosophila melanogaster (7).Despite the similarities between Argonaute proteins, AGO2 is the only human Argonaute protein believed to have the endonuclease activity (Slicer) due to a unique amino-terminal region (8-10), and Ago2 but not Ago1 gene deletions are embryonic lethal in mice (11). AGO2 was shown to promote transcriptional gene silencing in a microRNA (miRNA)-dependent manner (12) and to affect nucleosome occupancy at certain transcription start sites through interaction with the SWI/SNF complex (13). Furthermore, AGO1 has been shown to interact with actively transcribed genes and enhancer regions (14, 15). Recent work has identified expanded binding capabilities of AGO2, which include binding to longer RNAs such as pre-miRNAs (65 to 75 nucleotides [nt]) and full-length tRNAs (ϳ75 nt) (16)(17)(18)(19)(20). To wit, AGO2 was recently shown to functionally interact with DICER in human nuclei but was unable to load duplex small interfering RNA (siRNA), indicating that nuclear RNAi processes may proceed through a mechanism distinct from that observed in the cytoplasm (21). These findings indicate that AGO2 may have roles beyo...

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Section: Human Argonaute 2 Binds To Actively Transcribed Trna Genesmentioning

confidence: 99%

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confidence: 99%

Argonaute 2 Binds Directly to tRNA Genes and Promotes Gene Repression in cis

Woolnough

Atwood

Giles

2015

Molecular and Cellular Biology

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“…At residue 37, mutants lacking t 6 A 37 (N 6 -threonlycarbamoyladenosine) (El Yacoubi et al 2011;Srinivasan et al 2011) or m 1 G 37 grow very poorly , and lack of i 6 A 37 (N 6 -isopentenyladenosine) results in reduced nonsense suppression (Dihanich et al 1987) and tRNA gene-mediated silencing (Pratt-Hyatt et al 2013), as well as increased resistance to certain antifungal drugs (Suzuki et al 2012). Similarly, lack of Cm 32 and Nm 34 due to lack of TRM7 leads to poor growth because of reduced function of tRNA Phe (Pintard et al 2002;Guy et al 2012).…”

Section: Introductionmentioning

confidence: 99%

Functional importance of Ψ₃₈ and Ψ₃₉ in distinct tRNAs, amplified for tRNA^Gln(UUG) by unexpected temperature sensitivity of the s²U modification in yeast

Han

Kon

Phizicky

2014

RNA

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The numerous modifications of tRNA play central roles in controlling tRNA structure and translation. Modifications in and around the anticodon loop often have critical roles in decoding mRNA and in maintaining its reading frame. Residues U 38 and U 39 in the anticodon stem-loop are frequently modified to pseudouridine (Ψ) by members of the widely conserved TruA/Pus3 family of pseudouridylases. We investigate here the cause of the temperature sensitivity of pus3Δ mutants of the yeast Saccharomyces cerevisiae and find that, although Ψ 38 or Ψ 39 is found on at least 19 characterized cytoplasmic tRNA species, the temperature sensitivity is primarily due to poor function of tRNA Gln(UUG) , which normally has Ψ 38 . Further investigation reveals that at elevated temperatures there are substantially reduced levels of the s 2 U moiety of mcm 5 s 2 U 34 of tRNA Gln(UUG) and the other two cytoplasmic species with mcm 5 s 2 U 34 , that the reduced s 2 U levels occur in the parent strain BY4741 and in the widely used strain W303, and that reduced levels of the s 2 U moiety are detectable in BY4741 at temperatures as low as 33°C. Additional examination of the role of Ψ 38,39 provides evidence that Ψ 38 is important for function of tRNA Gln(UUG) at permissive temperature, and indicates that Ψ 39 is important for the function of tRNA Trp(CCA) in trm10Δ pus3Δ mutants and of tRNA Leu(CAA) as a UAG nonsense suppressor. These results provide evidence for important roles of both Ψ 38 and Ψ 39 in specific tRNAs, and establish that modification of the wobble position is subject to change under relatively mild growth conditions.

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“…The hypothesis that tRNA transcripts might directly inhibit Pol II transcription is particularly interesting because Pol II transcription is antagonized in the immediate vicinity of tRNA genes (Kinsey and Sandmeyer 1991;Hull et al 1994;Bolton and Boeke 2003). This local silencing of Pol II transcription by tRNA genes, termed tRNA gene-mediated (tgm) silencing, involves subnuclear clustering of the tRNA genes to the nucleolus (Wang et al 2005) mediated by condensin (Haeusler et al 2008) and modification of local chromatin structure Pratt-Hyatt et al 2013). In addition, pretRNA transcripts appear necessary for silencing, but their contribution to the silencing mechanism remains uncharacterized (Pratt-Hyatt et al 2013).…”

Section: Introductionmentioning

confidence: 99%

“…This local silencing of Pol II transcription by tRNA genes, termed tRNA gene-mediated (tgm) silencing, involves subnuclear clustering of the tRNA genes to the nucleolus (Wang et al 2005) mediated by condensin (Haeusler et al 2008) and modification of local chromatin structure Pratt-Hyatt et al 2013). In addition, pretRNA transcripts appear necessary for silencing, but their contribution to the silencing mechanism remains uncharacterized (Pratt-Hyatt et al 2013). Since tRNA and 5S rRNA genes are the only repetitive Pol III transcription units in yeast (there are no SINE elements in these small genomes), we initially set out to investigate the ability of these yeast transcripts to bind directly to purified Pol II from Saccharomyces cerevisiae and inhibit transcription.…”

Section: Introductionmentioning

confidence: 99%

RNAs nonspecifically inhibit RNA polymerase II by preventing binding to the DNA template

Pai

Kaplan

Kweon

et al. 2014

RNA

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Many RNAs are known to act as regulators of transcription in eukaryotes, including certain small RNAs that directly inhibit RNA polymerases both in prokaryotes and eukaryotes. We have examined the potential for a variety of RNAs to directly inhibit transcription by yeast RNA polymerase II (Pol II) and find that unstructured RNAs are potent inhibitors of purified yeast Pol II. Inhibition by RNA is achieved by blocking binding of the DNA template and requires binding of the RNA to Pol II prior to open complex formation. RNA is not able to displace a DNA template that is already stably bound to Pol II, nor can RNA inhibit elongating Pol II. Unstructured RNAs are more potent inhibitors than highly structured RNAs and can also block specific transcription initiation in the presence of basal transcription factors. Crosslinking studies with ultraviolet light show that unstructured RNA is most closely associated with the two large subunits of Pol II that comprise the template binding cleft, but the RNA has contacts in a basic residue channel behind the back wall of the active site. These results are distinct from previous observations of specific inhibition by small, structured RNAs in that they demonstrate a sensitivity of the holoenzyme to inhibition by unstructured RNA products that bind to a surface outside the DNA cleft. These results are discussed in terms of the need to prevent inhibition by RNAs, either though sequestration of nascent RNA or preemptive interaction of Pol II with the DNA template.

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Mod5 protein binds to tRNA gene complexes and affects local transcriptional silencing

Cited by 31 publications

References 56 publications

Argonaute 2 Binds Directly to tRNA Genes and Promotes Gene Repression in cis

Argonaute 2 Binds Directly to tRNA Genes and Promotes Gene Repression in cis

Functional importance of Ψ₃₈ and Ψ₃₉ in distinct tRNAs, amplified for tRNA^Gln(UUG) by unexpected temperature sensitivity of the s²U modification in yeast

RNAs nonspecifically inhibit RNA polymerase II by preventing binding to the DNA template

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Mod5 protein binds to tRNA gene complexes and affects local transcriptional silencing

Cited by 31 publications

References 56 publications

Argonaute 2 Binds Directly to tRNA Genes and Promotes Gene Repression in cis

Argonaute 2 Binds Directly to tRNA Genes and Promotes Gene Repression in cis

Functional importance of Ψ38 and Ψ39 in distinct tRNAs, amplified for tRNAGln(UUG) by unexpected temperature sensitivity of the s2U modification in yeast

RNAs nonspecifically inhibit RNA polymerase II by preventing binding to the DNA template

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Functional importance of Ψ₃₈ and Ψ₃₉ in distinct tRNAs, amplified for tRNA^Gln(UUG) by unexpected temperature sensitivity of the s²U modification in yeast