cap cliptm acid pyrophosphatase  (Cellscript Inc)

Journal: Nucleic Acids Research

Article Title: 3′ UTR-mediated regulation of a protein chaperone by the pspA mRNA in Streptococcus pneumoniae

doi: 10.1093/nar/gkag481

Figure Lengend Snippet: F5 is a pspA mRNA-derived RNA fragment. ( A ) The genetic region of F5 is conserved between the S. pneumoniae D39 and TIGR4 strains and is located directly downstream the pspA coding sequence (CDS) inside the 3′ UTR. ( B ) The distribution of pneumococcal 3′ UTR lengths reveal that the pspA 3′ UTR is longer than the majority of predicted 3′ UTRs in pneumococcus. UTR lengths were calculated based on transcript borders and CDS coordinates annotated in the D39V genome (GenBank: CP027540.1 ). ( C ) Using a F5 ( pspA -3′ UTR)-specific probe, the F5 RNA levels were analyzed by northern blot analysis using RNA from standard growth conditions (OD 600 0.1, 0.4, and 1.0 in C + Y media at 37°C), and different stress- and infection-relevant conditions (exponentially growing pneumococci were exposed to: 4 µg/ml antimicrobial peptide LL-37 for 30 min, 100 µg/ml ampicillin treatment for 30 min, competence induction with 0.4 µg/ml competence-stimulating peptide (CSP) for 12 min, sodium-chloride stress (0.5 M) for 30 min and heat-stress for 30 min at 42°C). Asteriks denote a fragment potentially corresponding to the ∼170 nt F5 RNA identified by Mann et al . . ( D ) Northern blot analysis shows a temperature-dependent generation of F5 RNA fragments. ( E ) The heat stress-induced F5 RNA fragment represents a processed transcript, as demonstrated by 5′ RACE analysis using Cap-Clip™ Acid Pyrophosphatase (CC-AP). Total RNA from heat-stressed D39 WT cultures was either treated with CC-AP to remove 5′ caps or left untreated. RNA samples were then ligated to an RNA adapter, reverse transcribed, and amplified using adapter-specific and F5-specific primers. The resulting products were resolved on an agarose gel to assess transcript processing. Asterisks indicate the fragment that was purified and sequenced. ( F ) The 5′-end of the heat stress generated F5 RNA is located exactly 20 nt downstream the pspA stop codon, matching the genetic coordinates identified by Mann et al . ( G ) Growth experiment with D39 WT and Δ 3UTR shows no effect of pspA -3′ UTR on growth management at different temperatures. Each data point represents mean of three biological replicates, with standard deviations as error bars.

Article Snippet: One part of the sample was treated with the Cap-ClipTM Acid Pyrophosphatase (10 U, Cellscript, C-CC15011H) for 30 min at 37°C, and the other was left untreated.

Techniques: Derivative Assay, Sequencing, Northern Blot, Infection, Reverse Transcription, Amplification, Agarose Gel Electrophoresis, Purification, Generated