A multiplicity of factors contributes to selective RNA polymerase III occupancy of a subset of RNA polymerase III genes in mouse liver

Abstract

The genomic loci occupied by RNA polymerase (RNAP) III have been characterized in human culture cells by genomewide chromatin immunoprecipitations, followed by deep sequencing (ChIP-seq). These studies have shown that onlỹ 40% of the annotated 622 human tRNA genes and pseudogenes are occupied by RNAP-III, and that these genes are often in open chromatin regions rich in active RNAP-II transcription units. We have used ChIP-seq to characterize RNAP-III-occupied loci in a differentiated tissue, the mouse liver. Our studies define the mouse liver RNAP-III-occupied loci including a conserved mammalian interspersed repeat (MIR) as a potential regulator of an RNAP-III subunit-encoding gene. They reveal that synteny relationships can be established between a number of human and mouse RNAP-III genes, and that the expression levels of these genes are significantly linked. They establish that variations within the A and B promoter boxes, as well as the strength of the terminator sequence, can strongly affect RNAP-III occupancy of tRNA genes. They reveal correlations with various genomic features that explain the observed variation of 81% of tRNA scores. In mouse liver, loci represented in the NCBI37/mm9 genome assembly that are clearly occupied by RNAP-III comprise 50 Rn5s (5S RNA) genes, 14 known non-tRNA RNAP-III genes, nine Rn4.5s (4.5S RNA) genes, and 29 SINEs. Moreover, out of the 433 annotated tRNA genes, half are occupied by RNAP-III. Transfer RNA gene expression levels reflect both an underlying genomic organization conserved in dividing human culture cells and resting mouse liver cells, and the particular promoter and terminator strengths of individual genes.[Supplemental material is available for this article.]RNA polymerase III (RNAP-III) synthesizes short RNAs involved in essential cellular processes, including protein synthesis, RNA maturation, and transcriptional control, but until recently, the full extent of the genomic loci occupied, and therefore probably transcribed, by RNAP-III in vivo, was unknown. Several groups have now used the ChIP-seq technique, i.e., chromatin immunoprecipitation followed by deep sequencing, to localize genome-wide RNAP-III and some of its transcription factors in several human cultured cell lines. These experiments have revealed a relatively modest number of new RNAP-III transcription units, from a few 10s to about 200, depending on the criteria applied (Barski et al. 2010;Canella et al. 2010;Moqtaderi et al. 2010;Oler et al. 2010). In addition, most previously known RNAP-III genes were occupied by RNAP-III. Thus, in such cells RNAP-III and some of its transcription factors occupied 17 RN5S loci annotated on chromosome 1 in the NCBI/hg18 genome assembly, the VTRNA1-1, VTRNA1-2, VTRNA1-3, and VTRNA2-1 (hsa-mir-886) genes coding for vault RNAs, three SRP genes, 14 SNAR genes, five RNU6 genes, the RNU6ATAC gene, the RN7SK, RMRP, and RPPH1 genes, and the RNY1, RNY3, RNY4, and RNY5 (hsa-mir-1975) genes. Noticeably, however, a large fraction of the annotated tRNA genes was devoid of...

High-resolution mapping of transcriptional dynamics across tissue development reveals a stable mRNA–tRNA interface

“…1B). Overall, the set of tRNA genes expressed during mouse development was similar to the tRNA genes identified as expressed in previous studies (Kutter et al 2011;Canella et al 2012;Carriere et al 2012;Renaud et al 2014). The overwhelming majority (93%, 290 of 311) of active tRNA genes resided in genomic clusters (Methods).…”

“…For each experiment at each tissue and developmental stage, we performed two biological replicates that were highly correlated Methods). This approach allowed us to quantify expression levels for protein-coding genes, as well as Pol III occupancy at every tRNA locus, which quantitatively captures the utilization of each tRNA gene (Barski et al 2010;Moqtaderi et al 2010;Oler et al 2010;Kutter et al 2011;Canella et al 2012;Carriere et al 2012;Renaud et al 2014).…”

“…We did this since prior studies have shown that sequence variation in internal regulatory sequences of tRNA genes have no clear relationship with their expression levels (Oler et al 2010;Canella et al 2012). We searched for regulatory sequence elements acting in cis that could direct the Pol III recruitment to tRNA genes as described previously (Giuliodori et al 2003;Bloom-Ackermann et al 2014).…”

“…gene usage and transcript abundance (Methods) (Barski et al 2010;Moqtaderi et al 2010;Oler et al 2010;Kutter et al 2011;Canella et al 2012;Carriere et al 2012;Renaud et al 2014). From E15.5 to P29 in liver and brain, 311 of the 433 tRNA genes predicted by tRNAscan-SE (Schattner et al 2005) were utilized (Supplemental Fig.…”

Gene duplication and neofunctionalization: POLR3G and POLR3GL

“…We first compared the results obtained for POLR3D in mouse liver with our previously published POLR3D results in the same tissue . As shown in Figure 3A, the correlation of scores for the tRNA genes and SINEs identified by Canella et al (2012) was extremely high (Spearman correlation coefficient 0.98), which is remarkable given that the two experiments were done at different times, by different people, and were sequenced on different machines (Illumina Genome Analyzer II in Canella et al [2010Canella et al [ , 2012 and Illumina HiSeq 2000 in this work). However, seven Rn5s (5S) and seven tRNA genes above the cutoff in Canella et al (2012) were below the cutoff in this study (note, however, that all but n-Tg4 [number 93] and n-Te18 [number 355] are above the cutoff in Hepa 1-6 cells) (indicated in yellow in column D of Supplemental Table S3) and reciprocally for 26 tRNA genes (indicated in light green in column D of Supplemental Table S3).…”