Analysis of pre-rRNAs in heat-shocked HeLa cells allows identification of the upstream termination site of human polymerase I transcription.

Abstract

Human rRNA precursors from normal or stressed HeLa cells were studied by Si nuclease mapping of unlabeled RNA and by antisense RNase mapping of RNA from cells that had been labeled in vivo with[32p]PO4. Heating cells to 43°C decreased the amount of newly synthesized rRNA to less than 5% of the control level and led to greater than 95% inhibition of transcription termination at a region 355 to 362 nucleotides downstream of the 3' end of 28S rRNA, with readthrough continuing into the next transcription unit. Heating of cells to 42°C led to 60% inhibition of termination at this site; 50% of transcripts that extended into the nontranscribed spacer ended in a region 200 to 210 nucleotides upstream of the polymerase I (Pol I) initiation site. This is presumed to be the human upstream transcription termination site because of the absence of RNAs with a 5' end corresponding to this region, the location relative to the Pol I initiation site (which is similar to the location of upstream terminators in other species), and the fact that it is 15 to 25 nucleotides upstream of the sequence GGGTTGACC, which has an 8-of-9 base identity with the sequence 3' of the downstream termination site. Surprisingly, treatment of cells with sodium arsenite, which also leads to the induction of a stress response, did not inhibit termination. Pol I initiation was decreased to the same extent as termination, which lends support to the hypothesis that termination and initiation are coupled. Although termination was almost completely inhibited at 43°C, the majority of the recently synthesized rRNAs were processed to have the correct 3' end of 28S. This finding suggests that 3'-end formation can involve an endonucleolytic cut and is not solely dependent on exonucleolytic trimming of correctly terminated rRNAs.

Duration of the first steps of the human rRNA processing

“…16 The human primary transcripts seem to be more stable (with the half-life about 3 min) than those of rodents. This supposition agrees with the data of earlier works 25,29,30 indicating that the first processing steps are more rapid in murine cells than in human cells.…”

“…In mammalian cells, ActD in concentration of 0.05 μg/ml inhibits pol I, but largely spares the activity of RNA polymerase II. 25,26 Here we examined the effects of this low ActD concentration on the expression levels of ETS-1 and ETS-2. HeLa and LEP cells were incubated with 0.05 μg/ml ActD for 10, 30, 50, 70, 90 and 120 min.…”

Analysis of the human immunodeficiency virus-1 RNA packageome

“…Some RNAs, such as U1 snRNAs that have not undergone 3 ′ cleavage, have only been detected previously when snRNP assembly was blocked with protein synthesis inhibitors (Yong et al 2010) or the catalytic subunit of the integrator complex was depleted (Baillat et al 2005). Other RNAs, such as transcripts from the rDNA IGS, have primarily been detected in response to environmental stressors such as heat-shock and acidosis (Labhart and Reeder 1987;Parker and Bond 1989;Audas et al 2012). Our finding that these unusual RNAs do not increase in virus-infected cells (Fig.…”

“…4C; Grummt et al 1985). Although Pol I normally terminates several hundred bp downstream from the 28S rRNA 3 ′ end, read-through transcripts extending through the IGS become prominent during heat shock and several other forms of stress (Labhart and Reeder 1987;Parker and Bond 1989;Audas et al 2012). Remarkably, in addition to many reads mapping to mature rRNAs, reads mapping throughout the IGS were detected in our virion data set but not in media from uninfected cells (Fig.…”

An RNA polymerase I termination site can stimulate the adjacent ribosomal gene promoter by two distinct mechanisms in Xenopus laevis.

“…Despite the lack of termination, we were unable to detect any decrease in transcription initiation under heat shock conditions. In cultured human cells, heat shock also leads to a failure of polymerase I termination and increased readthrough on the endogenous genes (Parker and Bond 1989). In the human case, however, a large decrease in transcription initiation was also detected, consistent with damage to the initiation complex.…”