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. 2018 Mar 14;7:e33684. doi: 10.7554/eLife.33684

Figure 5. GsrN co-purifies with multiple RNAs, including catalase/peroxidase katG mRNA.

(A) GsrN-target co-purification strategy. GsrN(black)-PP7hp(purple) fusions were expressed in a ΔgsrN background. PP7 RNA hairpin (PP7hp) inserted at nucleotide 37 (gsrN(37)::PP7hp) was used as the bait. PP7hp fused to the 3’ hairpin of gsrN (PP7hp::gsrN-3’)served as a negative control. Stationary phase cultures expressing these constructs were lysed and immediately flowed over an amylose resin column containing immobilized PP7hp binding protein (MBP-PP7cp-His). (B) GsrN-PP7hp purification from strains bearing gsrN(37)::PP7hp (left) and PP7hp::gsrN-3’ (right) was monitored by Northern Blot with probes complementary to 5’ end of GsrN and PP7hp, respectively. Lysate, flow through (FT), buffer wash, and elution fractions are blotted. Approximately 1 µg RNA was loaded per lane, except for buffer wash (insufficient amount of total RNA). (C) Annotation-based analysis of transcripts that co-purify with gsrN(37)::PP7hp (Figure 5—source data 1). Log10 reads per kilobase per million reads (RPKM) is plotted against the ln(-log10(false discovery rate corrected p-value)). Dashed red lines mark the enrichment co-purification thresholds. Genes enriched in the gsrN(37)::PP7hp purification compared to PP7hp::gsrN-3’ are blue; labels correspond to gene names or C. crescentus strain NA1000 CCNA GenBank locus ID. Data represent triplicate purifications of gsrN(37)::PP7hp and duplicate PP7hp::3’GsrN control purifications. Log adjusted p-values of zero are plotted as 10−260. (D) Sliding-window analysis of transcripts that co-purify with gsrN(37)::PP7hp (Figure 5—source data 2). Points represent 25 bp genome windows. RPKM values for each window were estimated by EDGE-pro; p-values were estimated by DESeq. Windows that map to genes identified in (C) are blue. Orange indicates windows with significant and highly abundant differences in mapped reads between gsrN(37)::PP7hp fractions and the PP7hp::gsrN-3’ negative control fractions. Dashed red lines denote cut-off value for windows enriched in the gsrN(37)::PP7hp fractions. Grey points within the dashed red lines are signal that mapped to rRNA. (E) Predicted loops in GsrN accessible for mRNA target base pairing are emphasized in colored texts. A putative mRNA target site complementary to a cytosine-rich tract in the 5’ GsrN loop is represented as a sequence logo. Similar logo was generated for the target site sequences complementary to the 2nd exposed region in the 3’ end of GsrN. Logo was generated from IntaRNA 2.0.2 predicted GsrN-binding sites in transcripts enriched in the gsrN(37)::PP7hp pull-down. 5’ binding motif is present in 32 of the transcripts identified in (C) and (D) and 3’ binding motif is present in 27 of the transcripts identified in (C) and (D). (F) Density of reads mapping to katG that co-purified with gsrN(37)::PP7hp (blue) and PP7hp::gsrN-3’ (red). Read density in each dataset represents read coverage at each nucleotide divided by the number of million reads mapped in that data set. Data represent mean ±SD of three replicate gsrN(37)::PP7hp and two replicate PP7hp::gsrN-3’ purifications.

Figure 5—source data 1. Excel file of the output from Rockhopper analysis (Tjaden, 2015) on the RNA-Seq samples from the PP7 affinity purified total RNA samples.
Figure 5C can be created using the python and R scripts in https://github.com/mtien/Sliding_window_analysis.
elife-33684-fig5-data1.xlsx (856.7KB, xlsx)
DOI: 10.7554/eLife.33684.015
Figure 5—source data 2. Zipped file contain three files.
These files include the sliding window analysis files generated from mapping the reads from the RNA-Seq experiment of the PP7 affinity purified total RNA samples. Figure 5D can be created using the scripts in https://github.com/mtien/Sliding_window_analysis.
elife-33684-fig5-data2.zip (1.6MB, zip)
DOI: 10.7554/eLife.33684.016
Figure 5—source data 3. FASTA file that contains the windows of enrichment and total gene sequences of genes identified in the PP7 affinity purified total RNA samples.
elife-33684-fig5-data3.txt (13.8KB, txt)
DOI: 10.7554/eLife.33684.017

Figure 5.

Figure 5—figure supplement 1. Identification, purification and biochemical characterization of GsrN-PP7hp chimeras.

Figure 5—figure supplement 1.

(A) Predicted GsrN secondary structure diagram from mFold (Zuker, 2003). Cyan and pink represent the 5’ and 3’ products, respectively, determined by primer extension and northern blot analyses (Figure 3). Numbered positions along the secondary structure indicate where PP7 RNA hairpin sequences (PP7hp) were inserted into gsrN.. (B) Wild type, ΔgsrN-EV, and ΔgsrN +gsrN-PP7hp strains were subjected to hydrogen peroxide, diluted, and titered as in Figure 1—figure supplement 2B. Empty vector (EV) strains carry the integrating plasmid pMT552. The nucleotide position of each PP7hp insertion in gsrN is marked above each bar. Data represent mean ±SD of three independent trials. (C) Northern blot of total RNA from stationary phase cultures (OD660 ≈ 1.0, a condition in which GsrN levels were observed to be the highest) of ΔgsrN strains carrying gsrN-PP7hp fusions. The blot was probed with oligonucleotides complementary to the 5’ and to the 3’ ends of GsrN. Blot is overexposed to reveal minor products. Dark blue boxes mark full length GsrN, cyan boxes mark 5’ isoforms, and pink boxes mark 3’ isoforms. (D) Northern blot of same samples as in (C) run in parallel and probed with oligonucleotides complementary to the PP7 hairpin sequence.