Journal: Nucleic Acids Research
Article Title: The complete pathway for co-transcriptional mRNA maturation within a large protein of a non-segmented negative-strand RNA virus
doi: 10.1093/nar/gkae659
Figure Lengend Snippet: Nascent transcripts of 21–23 nt, but not of 13–15 nt, are capped with fast-transcribing rL. ( A ) N-RNA was pre-incubated with rL and rP in the presence of ATP, CTP, and SAM. After adding saturating concentrations of UTP, GTP, and [α- 32 P]GDP, the reaction mixtures were incubated for the indicated periods. Cap-labeled transcripts were analyzed by 20% urea–PAGE followed by autoradiography. Marker RNAs are as follows: i, a mixture of 5′-capped oligo-RNAs with the VSV N mRNA start sequence (10 nt, Gppp N 10 ; 20 nt, Gppp N 20 ; 30 nt, Gppp N 30 ); ii, a hydroxide cleavage ladder of Gppp N 30 , 3′-dephosphorylated. The positions of the gel origin (ori.) and bottom (bot.) are indicated. ( B ) The transcripts of 21–23 nt synthesized in the presence of [α- 32 P]GDP for 60 s (see panel A, lane 4) were purified by urea–PAGE and digested with Nase P1. The digests were analyzed as in Figure . ( C ) Nascent transcripts were pulse-labeled with [α- 32 P]GDP for 60 sec as in panel A. After adding excess cold GDP, cap-labeled transcripts were chased for the indicated periods, and analyzed as in panel A. Maker lane iii indicates Ambion Decade Markers. ( D ) Cap-labeled nascent transcripts before and after chasing for 180 s (see panel C, lanes 1 and 4) were digested with Nase P1. The digests were analyzed as in Figure . ( E ) Nascent transcripts were synthesized in the presence of [α- 32 P]GDP (left scheme) or cold GDP (right scheme) for 60 s as in panel A. The latter RNAs were post-transcriptionally capped with [α- 32 P]GDP by rL. ( F ) Co-transcriptionally (lane 1) or post-transcriptionally (lane 2) capped transcripts were analyzed as in panel A. Marker RNAs are as follows: iv, a mixture of Gppp N 10 and Gppp N 20 ; v, a hydroxide cleavage ladder of Gppp N 20 , 3′-dephosphorylated.
Article Snippet: 32 P-Labeled Ambion Century-Plus and Decade RNA markers (Life Technologies/Thermo Fisher Scientific) were prepared according to the manufacturer's protocols.
Techniques: Incubation, Labeling, Autoradiography, Marker, Sequencing, Synthesized, Purification
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![Nascent transcripts of 21–23 nt, but not of 13–15 nt, are capped with fast-transcribing rL. ( A ) <t>N-RNA</t> was pre-incubated with rL and rP in the presence of ATP, CTP, and SAM. After adding saturating concentrations of UTP, GTP, and [α- 32 P]GDP, the reaction mixtures were incubated for the indicated periods. Cap-labeled transcripts were analyzed by 20% urea–PAGE followed by autoradiography. Marker RNAs are as follows: i, a mixture of 5′-capped oligo-RNAs with the VSV N mRNA start sequence (10 nt, Gppp N 10 ; 20 nt, Gppp N 20 ; 30 nt, Gppp N 30 ); ii, a hydroxide cleavage ladder of Gppp N 30 , 3′-dephosphorylated. The positions of the gel origin (ori.) and bottom (bot.) are indicated. ( B ) The transcripts of 21–23 nt synthesized in the presence of [α- 32 P]GDP for 60 s (see panel A, lane 4) were purified by urea–PAGE and digested with Nase P1. The digests were analyzed as in Figure . ( C ) Nascent transcripts were pulse-labeled with [α- 32 P]GDP for 60 sec as in panel A. After adding excess cold GDP, cap-labeled transcripts were chased for the indicated periods, and analyzed as in panel A. Maker lane iii <t>indicates</t> <t>Ambion</t> Decade Markers. ( D ) Cap-labeled nascent transcripts before and after chasing for 180 s (see panel C, lanes 1 and 4) were digested with Nase P1. The digests were analyzed as in Figure . ( E ) Nascent transcripts were synthesized in the presence of [α- 32 P]GDP (left scheme) or cold GDP (right scheme) for 60 s as in panel A. The latter RNAs were post-transcriptionally capped with [α- 32 P]GDP by rL. ( F ) Co-transcriptionally (lane 1) or post-transcriptionally (lane 2) capped transcripts were analyzed as in panel A. Marker RNAs are as follows: iv, a mixture of Gppp N 10 and Gppp N 20 ; v, a hydroxide cleavage ladder of Gppp N 20 , 3′-dephosphorylated.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1362/pmc11381362/pmc11381362__gkae659fig2.jpg)
![L methylates the cap on pre-mRNAs during mRNA chain elongation. ( A ) Diagrams of each pulse-chase experiment are shown. ( B and C ) After preincubation of N-RNA, rL, and rP in the presence of ATP, CTP, and SAM, nascent transcripts were pulse-labeled with [α- 32 P]GDP in the presence of the limiting concentration of GTP. After adding excess concentrations of cold GDP and GTP, cap-labeled nascent transcripts were chased for the indicated periods (10 sec to 11 min) (see panel A, middle) and analyzed by 20% (B) or 5% (C) urea–PAGE followed by autoradiography. An asterisk indicates an abortive transcript of 40 nt. Marker RNAs are as follows: viii, Ambion Century-Plus RNA Markers, ix, deadenylated VSV N , P / M , and G mRNAs. ( D ) Cap-labeled transcripts chased for 11 min (lane 1) were digested with RNase H in the presence of oligo(dT) (lane 2) and analyzed as in panel C. ( E ) Cap-labeled transcripts chased for the indicated periods were digested with Nase P1. The digests were analyzed as in Figure . ( F ) Pulse-chase reactions performed in the absence of SAM (see panel A, lower). During the chase period, the reactions were received water or SAM (final concentration: 30 μM) at the indicated time point and further incubated for 3 min. The cap methylation status was analyzed as in panel E. ( G – I ) Chase reactions were terminated at the indicated time point and subjected to ultracentrifugation to separate free transcripts (Supernatants, Sup) from RNP-associated transcripts (Precipitates, Ppt). Transcripts in each fraction were analyzed by 20% (H) or 5% (I) urea–PAGE followed by autoradiography.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_1362/pmc11381362/pmc11381362__gkae659fig5.jpg)