No abstract

Transcription can be quite disruptive for chromatin so cells have evolved mechanisms to preserve chromatin integrity during transcription, thereby preventing the emergence of cryptic transcripts from spurious promoter sequences. How these transcripts are regulated and processed remains poorly characterized. Notably, very little is known about the termination of cryptic transcripts. Here, we used RNA-Seq to identify and characterize cryptic transcripts in Spt6 mutant cells (spt6-1004) in Saccharomyces cerevisiae. We found polyadenylated cryptic transcripts running both sense and antisense relative to genes in this mutant. Cryptic promoters were enriched for TATA boxes, suggesting that the underlying DNA sequence defines the location of cryptic promoters. While intragenic sense cryptic transcripts terminate at the terminator of the genes that host them, we found that antisense cryptic transcripts preferentially terminate near the 3΄-end of the upstream gene. This finding led us to demonstrate that most terminators in yeast are bidirectional, leading to termination and polyadenylation of transcripts coming from both directions. We propose that S. cerevisiae has evolved this mechanism in order to prevent/attenuate spurious transcription from invading neighbouring genes, a feature that is particularly critical for organisms with small compact genomes.

Highlights d Tyr1 regulates termination of some coding genes and of most non-coding transcripts d Tyr1 mediates a pausing event in the 5 0 end of genes d Tyr1-mediated pausing is required for efficient NNSdependent termination d Nrd1 and Sen1 mediate termination independently of their ability to bind the CTD

The enzyme UGT2B7 is one of the most active UDP-glucuronosyltransferases (UGTs) involved in drug metabolism and in maintaining homeostasis of endogenous compounds. We recently reported the existence of 22 UGT2B7 mRNAs, two with a classic 59 region but alternative 39 ends namely UGT2B7_v5 (containing a novel terminal exon 6b) and _v7 (exon 5 excluded) that encode enzymatically inactive isoforms 2 and 4 (i2 and i4), respectively. The v1 mRNA encoding the UGT2B7 enzyme (renamed isoform 1 or i1) is coexpressed with the splice variants v5 and v7 in human liver, kidney, and small intestine and the hepatic cell lines HepG2 and C3A. The presence of alternate v5 and v7 transcripts in isolated polysomes from these hepatic cells further supports endogenous protein translation. Cellular fractionation of clonal HEK293 cell lines overexpressing UGT2B7 isoforms demonstrates that i1, i2, and i4 proteins colocalize in the microsomal/Golgi fraction, whereas i2 and i4 can also be found in the cytosol; a finding sustained by immunofluorescence experiments using tagged proteins. By modifying splice variant abundance in overexpression in HEK293 and HepG2 cells as well as RNA interference experiments in HepG2 and C3A cells, we observe drug glucuronidation phenotypes compatible with variant-mediated repression of UGT2B7 activity without consequent alteration of the apparent enzyme affinity (K m ). Finally, coimmunoprecipitation experiments support a direct proteinprotein interaction of i2 and i4 proteins with the functional UGT2B7 enzyme as a potential causative mechanism. These findings point toward a novel autoregulatory mechanism of the UGT2B7 glucuronidation pathway by naturally occurring alternative i2 and i4 proteins.

Evidence for temporal variations in the nephrotoxicity of low doses of aminoglycosides were recently shown by using specific and sensitive parameters of renal toxicity. The aim of the present study was to evaluate the effect of a short period of fasting on the temporal variations in the renal toxicity of gentamicin. Twenty-eight normally fed (i.e., food and water were available ad libitum throughout the experiment) female Sprague-Dawley rats (weight, 175 to 220 g) and 28 fasted rats (i.e., only water was available during a 12-h fast before and a 24-h fast after gentamicin injection) were used. The animals were synchronized on a 14-h light, 10-h dark cycle (lights on at 0600 h) for 1 week before gentamicin administration. In July 1993, each group of animals was treated with a single intraperitoneal injection of saline (NaCl, 0.9%) or gentamicin (150 mg/kg of body weight) at either the peak (1400 h) or the trough (0200 h) of the previously determined toxicity. On day 1, the 24-h urinary excretion of beta-galactosidase, N-acetyl-beta-D-glucosaminidase, and gamma-glutamyltransferase was significantly higher in normally fed animals treated with gentamicin at 1400 h than in their time-matched controls and in normally fed animals treated at 0200 h (P < 0.01), which had normal levels of these enzymes. By contrast, the urinary excretion of these enzymes was significantly higher in both groups of gentamicin-treated, fasted rats than in their time-matched control groups (P < 0.01), reaching levels similar to those measured in normally fed rats treated at 1400 h. The accumulation of gentamicin was significantly lower in the renal cortex of normally fed rats treated at 0200 h than in rats treated at 1400 h (P < 0.05), but this time-dependent difference was not found in fasted rats treated at 0200 and 1400 h. Immunogold labeling done on ultrathin sections and observed by electron microscopy showed a similar subcellular localization of gentamicin in normally fed and fasted rats treated at either 1400 or 0200 h. These results suggest that the feeding period is of crucial importance in the temporal variations of the nephrotoxicity of gentamicin in rats.