Journal: eLife

Article Title: The final step of 40S ribosomal subunit maturation is controlled by a dual key lock

doi: 10.7554/eLife.61254

Figure Lengend Snippet: ( a ) Diagram representing steps of the pre-40S rRNA digestion by RNase H. ( b ) RNase H assays were performed on RNAs extracted from pre-40S particles purified with the mentioned StHA-tagged bait and separated on a 12% polyacrylamide gel. The 18S rRNA and its precursors were revealed by the 3′18S radiolabeled probe. Bands are separated with single nucleotide resolution, as shown in . ( c ) Signals corresponding to the 18S-E and 18S rRNAs were quantified by phosphorimaging and represented by the 18S/(18S + 18S-E) ratio for the different purified pre-40S particles. The average of three independent experiments is shown, with the standard deviation indicated on top of the histogram.

Article Snippet: After annealing by cooling down to room temperature for 20 min, the reaction mixture was diluted to 30 µL with a reaction mix containing 1X RNase H reaction buffer, 25 µM DTT, 0.5 U/l RNasin (Promega), and 50 U RNase H (New England Biolabs), and incubated at 37°C for 30 min.

Techniques: Purification, Standard Deviation

Journal: eLife

Article Title: The final step of 40S ribosomal subunit maturation is controlled by a dual key lock

doi: 10.7554/eLife.61254

Figure Lengend Snippet: Comparison of RNAse H digestion and alkaline hydrolysis assays shows nucleotide resolution between bands. (Left) RNase H digestion of rRNAs from pre-40S particles purified using a HASt-tagged version of LTV1 as bait (HASt-LTV1). (Right) Alkaline hydrolysis (OH− ladder) of an RNA molecule containing the 18S-ITS1 sequence recognized by the 3′18S probe at its 5′ end (see ). The samples were fractionated on a 12% polyacrylamide gel, and northern blot was probed with the 3′18S probe.

Article Snippet: After annealing by cooling down to room temperature for 20 min, the reaction mixture was diluted to 30 µL with a reaction mix containing 1X RNase H reaction buffer, 25 µM DTT, 0.5 U/l RNasin (Promega), and 50 U RNase H (New England Biolabs), and incubated at 37°C for 30 min.

Techniques: Purification, Sequencing, Northern Blot

Journal: eLife

Article Title: The final step of 40S ribosomal subunit maturation is controlled by a dual key lock

doi: 10.7554/eLife.61254

Figure Lengend Snippet: HEK cell lines expressing tagged version of LTV1, the catalytically inactive RIO1-D324A (RIO1 (kd)) or wild-type RIO1 (RIO1(wt)) were treated with scramble or RPS26 siRNAs for 48 hr. ( a ) RNase H assays were conducted as in on rRNAs of pre-40S particles purified with the mentioned StHA-tagged bait, either from RPS26-depleted or from control cells (scramble siRNA). ( b ) Signals corresponding to the 18S-E and 18S rRNA detected in ( a ) were quantified and represented as the 18S/(18S + 18S-E) ratio for the different pre-40S particles. Error bars, s.d. (n = 3) ( c ) Cell extracts and purified particles were analyzed by western blot using the indicated antibodies. ( d ) Bands corresponding to DIM2 and NOB1 (in the eluates) were quantified, corrected for pre-40S particle loading (using RPS19) and normalized to the control condition (set to 1). Error bars, s.d. (n = 3).

Article Snippet: After annealing by cooling down to room temperature for 20 min, the reaction mixture was diluted to 30 µL with a reaction mix containing 1X RNase H reaction buffer, 25 µM DTT, 0.5 U/l RNasin (Promega), and 50 U RNase H (New England Biolabs), and incubated at 37°C for 30 min.

Techniques: Expressing, Purification, Western Blot

Journal: eLife

Article Title: The final step of 40S ribosomal subunit maturation is controlled by a dual key lock

doi: 10.7554/eLife.61254

Figure Lengend Snippet: HEK cell lines expressing tagged versions of wild-type RIO1 ‘RIO1(wt)’ or of the catalytically inactive ‘RIO1(kd)’ were treated with RPS26 siRNAs for 48 hr to enrich particles in state A. Pre-40S particles were purified and incubated for 1 hr in buffer alone (mock condition) or in the presence of either 1 mM ATP, 1 mM AMP-PNP, 2 µg of RPS26, or 2 µg of RPS26 plus 1 mM ATP. ( a ) RNAse H assays were performed on the RNAs extracted from the particles. ( b ) The variation of cleavage efficiency upon addition of ATP or AMP-PNP is indicated by the 18S/(18S + 18S-E) ratio. The data correspond to five independent experiments. Statistical analysis was performed with a unilateral paired Wilcoxon test (‘sample greater than mock’) indicating p-values of 0.031 for all samples. ( c ) The variation of cleavage efficiency upon addition of ATP and/or RPS26 is indicated by the 18S/(18S + 18S-E) ratio. The data correspond to three to six independent measurements for each point. Statistical analysis was performed with a unilateral unpaired Wilcoxon test (‘sample greater than mock’ or ‘+RPS26+ATP greater than +RPS26’).

Article Snippet: After annealing by cooling down to room temperature for 20 min, the reaction mixture was diluted to 30 µL with a reaction mix containing 1X RNase H reaction buffer, 25 µM DTT, 0.5 U/l RNasin (Promega), and 50 U RNase H (New England Biolabs), and incubated at 37°C for 30 min.

Techniques: Expressing, Purification, Incubation