Nascent RNA signaling to yeast RNA Pol II during transcription elongation

<div><p>Transcription as the key step in gene expression is a highly regulated process. The speed of transcription elongation depends on the underlying gene sequence and varies on a gene by gene basis. The reason for this sequence dependence is not known in detail. Recently, our group studied the cross talk between the nascent RNA and the transcribing RNA polymerase by screening the <i>Escherichia coli</i> genome for RNA sequences with high affinity to RNA Pol by performing genomic SELEX. This approach led to the identification of <u>R</u>NA polymerase-binding <u>AP</u>tamers termed “RAPs”. RAPs can have positive and negative effects on gene expression. A subgroup is able to downregulate transcription via the activity of the termination factor Rho. In this study, we used a similar SELEX setup using yeast genomic DNA as source of RNA sequences and highly purified yeast RNA Pol II as bait and obtained almost 1300 yeast-derived RAPs. Yeast RAPs are found throughout the genome within genes and antisense to genes, they are overrepresented in the non-transcribed strand of yeast telomeres and underrepresented in intergenic regions. Genes harbouring a RAP are more likely to show lower mRNA levels. By determining the endogenous expression levels as well as using a reporter system, we show that RAPs located within coding regions can reduce the transcript level downstream of the RAP. Here we demonstrate that RAPs represent a novel type of regulatory RNA signal in <i>Saccharomyces cerevisiae</i> that act in <i>cis</i> and interfere with the elongating transcription machinery to reduce the transcriptional output.</p></div

RAP host genes display intragenic termination.

<p>(A) and (C) show a gene centered view on RNA Pol II occupancy at RAP-containing genes and genes without RAPs determined by available NET-Seq (A) and CRAC-Seq (C) data sets. To avoid signal distortion by the RNA Pol II 5’ peak, the first 200nts (and for symmetry reasons also the last 200nts) were excluded from the analysis. The remaining gene body was binned into ten equally spaced segments (deciles) and the upstream/downstream ratio of RNA Pol II occupancy was calculated for each segment. Distribution of ratios from segments harboring a RAP are separately visualized (blue bars) and show a consistently higher upstream RNA Pol II density. (B) and (D) Ratio of RNA Pol II occupancy between genic regions upstream and downstream of RAPs tested by NET-seq (B) and CRAC-seq (D). For each gene position upstream and downstream of a RAP the number of reads per position from each experiment was deduced and tested for a significant increase, decrease, or no significant change between the upstream and downstream region coverage (Poisson test; p-value threshold < 0.01). To estimate an expected background distribution accounting for the general decrease of Pol II occupancy along the gene body, the analysis was repeated with 100 randomized positions, conserving the relative RAP positions within the harboring gene. Both data sets, NET-Seq (B) and CRAC-Seq (D), show that RAP containing genes more often display a significant decrease downstream of the RAP compared to the randomized background.</p

Expression profiles of RAP-containing genes.

<p>(A) to (J) Transcript levels of 10 selected target genes (<i>MOT3</i>, <i>CYC8</i>, <i>CBK1</i>, <i>SSL2</i>, <i>PUF3</i>, <i>TOP3</i>, <i>TUP1</i>, <i>BUD8</i>, <i>MSI1</i> and <i>SSM4</i> respectively). Yeast cells were grown to exponential phase, total RNA was extracted and DNAseI digested. Reverse transcription was performed, and expression levels were quantified by qRT-PCR using amplicons upstream and downstream of RAPs as indicated in the respective figures. Values were normalized to the first 5’ amplicon and represent means ° SD n ≤ 3; ***p<0.001; **p<0.01; *p≤0.05; ns, not significant (tested by t-test).</p

Genomic SELEX RNA Pol II binding RNAs.

<p>(A) Schematic outline for our approach to isolate novel RNA Pol II-binding RNAs. After construction of a genomic library via random priming, DNA fragments are <i>in vitro</i> transcribed and subjected to multiple rounds of SELEX to isolate RNA Pol II-binding RNAs. (B) Enrichment profile for the selection of RNA Pol II-binding RNAs, showing the percentage of recovered RNA per cycle. RNAs were selected via filter binding. Low stringency conditions (10μM RNA and 1μM protein) were applied for the six consecutive cycles to firmly establish a population of binding species. Since a 10-fold molar excess of RNA over protein was used to support competition, these values correspond to 100% of the total amount of RNA able to bind RNA Pol II, assuming a single binding site on the protein. (C) After deep sequencing, the enriched RNA domains (~1300), termed RAPs (RNA polymerase-binding aptamers) were mapped to the yeast genome and classified according to their genomic location. Percentage of RAPs classified as being intergenic, sense or anti-sense to annotated genes, compared to the relative occurrence of these classes in the yeast genome. (D) Analysis of expression levels of RAP-containing genes. Only RAPs oriented in sense to open reading frames were analyzed (total 370). On the x-axis the genes are ordered according to their transcript abundance (TPM = transcripts per million) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194438#pone.0194438.ref062" target="_blank">62</a>]. On the y-axis the cumulative count of genes including a RAP up to the corresponding TPM is plotted. If RAPs were uniformly distributed over all genes irrespective of their transcript abundance the curve should follow the straight diagonal. Genes in segments with a reduced slope are depleted of RAPs, genes in segments with a larger slope than the diagonal are enriched in RAPs. Different segments were tested for a significant difference in RAP density to the expected uniform distribution using a Poisson test. (E) Bar graph showing the number of genes in each category and the percentage of RAPs in each category. (F) Analysis for overrepresented sequence motifs in RAPs compared to the genomic background. The three best ranked motifs according to their p-values are shown.</p

Isolation of small RNA-binding proteins from E. coli: evidence for frequent interaction of RNAs with RNA polymerase.

Bacterial small RNAs (sRNAs) are non-coding RNAs that regulate gene expression enabling cells to adapt to various growth conditions. Assuming that most RNAs require proteins to exert their activities, we purified and identified sRNA-binding factors via affinity chromatography and mass spectrometry. We consistently obtained RNA polymerase betasubunit, host factor Hfq and ribosomal protein S1 as sRNA-binding proteins in addition to several other factors. Most importantly, we observed that RNA polymerase not only binds several sRNAs but also reacts with them, both cleaving and extending the RNAs at their 3' ends. The fact that the RNA polymerase reacts with sRNAs maps their interaction site to the active centre cleft of the enzyme and shows that it takes RNAs as template to perform RNA-dependent RNA polymerase activity. We further performed genomic SELEX to isolate RNA polymerase-binding RNAs and obtained a large number of E. coli sequences binding with high affinity to this enzyme. In vivo binding of some of the RNAs to the RNA polymerase was confirmed via co-immunoprecipitation in cell extracts prepared from different growth conditions. Our observations show that RNA polymerase is able to bind and react with many different RNAs and we suggest that RNAs are involved in transcriptional regulation more frequently than anticipated

Nascent RNA signaling to yeast RNA Pol II during transcription elongation

Transcription as the key step in gene expression is a highly regulated process. The speed of transcription elongation depends on the underlying gene sequence and varies on a gene by gene basis. The reason for this sequence dependence is not known in detail. Recently, our group studied the cross talk between the nascent RNA and the transcribing RNA polymerase by screening the Escherichia coli genome for RNA sequences with high affinity to RNA Pol by performing genomic SELEX. This approach led to the identification of RNA polymerase-binding APtamers termed “RAPs”. RAPs can have positive and negative effects on gene expression. A subgroup is able to downregulate transcription via the activity of the termination factor Rho. In this study, we used a similar SELEX setup using yeast genomic DNA as source of RNA sequences and highly purified yeast RNA Pol II as bait and obtained almost 1300 yeast-derived RAPs. Yeast RAPs are found throughout the genome within genes and antisense to genes, they are overrepresented in the non-transcribed strand of yeast telomeres and underrepresented in intergenic regions. Genes harbouring a RAP are more likely to show lower mRNA levels. By determining the endogenous expression levels as well as using a reporter system, we show that RAPs located within coding regions can reduce the transcript level downstream of the RAP. Here we demonstrate that RAPs represent a novel type of regulatory RNA signal in Saccharomyces cerevisiae that act in cis and interfere with the elongating transcription machinery to reduce the transcriptional output.© 2018 Klopf et a