Functional interactions of RNA-capping enzyme with factors that positively and negatively regulate promoter escape by RNA polymerase II

Abstract

Capping of the 5' ends of nascent RNA polymerase II transcripts is the first pre-mRNA processing event in all eukaryotic cells. Capping enzyme (CE) is recruited to transcription complexes soon after initiation by the phosphorylation of Ser-5 of the carboxyl-terminal domain of the largest subunit of RNA polymerase II. Here, we analyze the role of CE in promoter clearance and its functional interactions with different factors that are involved in promoter clearance. FCP1-mediated dephosphorylation of the carboxyl-terminal domain results in a drastic decrease in cotranscriptional capping efficiency but is reversed by the presence of DRB sensitivity-inducing factor (DSIF). These results suggest involvement of DSIF in CE recruitment. Importantly, CE relieves transcriptional repression by the negative elongation factor, indicating a critical role of CE in the elongation checkpoint control mechanism during promoter clearance. This functional interaction between CE and the negative elongation factor documents a dynamic role of CE in promoter clearance beyond its catalytic activities.

Fig. 1.

Cotranscriptional capping in stalled transcription complexes. (A) Ternary complexes were incubated with 12 ng of CE and 50 μM GTP at 30°C. Capping reactions (lanes 1-5 for +16, lanes 6-10 for +24, and lanes 11-15 for +42) were stopped at the times shown. Products were subjected to phenol/chloroform extraction, ethanol precipitation, and PAGE/8 M urea, followed by autoradiography. The two small spots in lane 6 (above +24) are RNAs produced by slippage of RNAPII (30). (B) To confirm cap formation on RNA, +24 stalled complexes were incubated with [α-³²P]GTP with and without CE (120 ng). RNAs (24 nt) were isolated, treated with P1 nuclease and AP, and analyzed by TLC. Lane 1, untreated RNA; lanes 2 and 3, capped and uncapped RNAs digested with P1 nuclease and AP. The position of authentic GpppA run in parallel is indicated.

Fig. 2.

DSIF stimulates cotranscriptional capping. (A) Stalled +24 complexes were incubated alone (lane 1) or with increasing amounts of CE and GTP for 5 min (lanes 2-4). In parallel, +24 complexes that were first incubated with 10, 30, or 90 ng of DSIF for 10 min were further incubated with GTP in the absence (lanes 11-13) and presence (lanes 5-10) of CE for 5 min. (B) Quantitation of A.

Fig. 3.

FCP1 inhibits capping. (A) Ternary +24 complexes were incubated alone (lane 1) or with 0.06, 0.12, and 0.6 ng of CE and GTP for 5 min (lanes 2-4). In parallel, +24 complexes were first incubated with 60 ng of FCP1 for 30 min at 30°C, washed with TB500, and then equilibrated with TB60. Finally, FCP1 treated complexes were incubated with 0.06, 0.12, and 0.6 ng of CE (lanes 5-7) and GTP for an additional 5 min. Lane 8: complexes treated with FCP1 only. (B) Quantitation of A. (C) Ternary +24 complexes were preincubated with FCP1 or catalytically inactive mutant FCP1a for 30 min at 30°C and then treated with 12 ng of CE and GTP for an additional 5 min.

Fig. 4.

DSIF can overcome FCP1-mediated capping inhibition. (A) Ternary +24 complexes alone (lane 1) or incubated with 0.6 ng of CE and GTP for 5 min at 30°C (lane 2). Ternary complexes were incubated with 60 ng of FCP1 for 30 min at 30°C, washed to remove FCP1, resuspended in TB60, and treated with CE and GTP in the absence (lane 3) or presence (lanes 4-6) of varying amounts of DSIF. As control, complexes not treated with FCP1 were incubated with DSIF and CE (lanes 7-8). (B and C) Capping reactions in A were subjected to 6% SDS/PAGE and analyzed by Western blot with GAL4-CTD polyclonal antibody, which recognizes both phosphorylated (IIO) and nonphosphorylated (IIA) Rpb1 and monoclonal antibody H14 to detect specifically Ser-5 phosphorylated CTD. (D) Quantitation of A. (E) Ternary +24 complexes were treated with FCP1 for 30 min, and treated and untreated complexes were incubated with 0.6 ng CE in the absence and presence of 30 ng of DSIF. Reactions were stopped at the indicated times, and the percentage of capped RNA was determined by SDS/PAGE and autoradiography.

Fig. 5.

CE can overcome NELF-mediated transcription repression. (A) PICs were assembled with purified GTFs on a linear DNA by incubation in TB60 for 20 min at 30°C. DSIF (30 ng), NELF, and CE were then added as shown, and incubations continued for 10 min. Transcription reactions were chased with a mixture of 600 μM ATP, CTP, and GTP; 0.3 μM [α-³²P]UTP; 2 μM UTP and 16 units of RNasin for 6 min at 30°C. Reactions were stopped with stop buffer, extracted with phenol-chloroform, ethanol precipitated, and analyzed by 7% PAGE/8 M urea and autoradiography. PICs were chased with either NTPs alone (lane 1) or first incubated with DSIF (lane 2) or NELF (lane 3) or both (lane 4) and chased with NTPs. PICs incubated with both DSIF and NELF were further incubated with 0.12, 12, or 120 ng of CE (lanes 5-7) and chased with NTPs. For lanes 8 and 9, PICs were chased with NTPs in the presence of CE alone (12 and 120 ng). The dots indicate the approximate positions of transcripts of 42 and ≈200 nt. (B)Asin A, except that transcription reactions were chased with NTPs (50 μM ATP/5 μM CTP/0.3 μM [α-³²P]UTP/50 μM 3′-O^meGTP/1 μM UTP/16 units of RNasin) for 5 min to produce +24 stalled transcripts, which were analyzed by 18% PAGE in 8M urea.

Fig. 6.

Reversal of NELF-mediated transcription repression depends on CE but not on cap formation. (A) Western blot of K294A mutant CE and wild-type CE. (B) Guanylylation of CE. Wild-type and mutant CE were incubated in 20 μl of GTP-labeling buffer (25 mM Tris·HCl, pH 7.5/5 mM MgCl₂/0.5 mM DTT/10 μCi (1 Ci = 37 GBq) [α-³²P]GTP/0.1 μg of inorganic pyrophosphatase) at 37°C for 10 min and then analyzed by SDS/PAGE and autoradiography. (C) PICs were incubated with DSIF and NELF as in Fig. 5A and then chased with NTPs in the absence and presence of wild-type CE (12 and 120 ng, compare lanes 4-7), K294A inactive mutant CE (12 and 120 ng, compare lanes 8-10), or BSA as control (120 ng, compare lanes 11 and 12). Analysis by 7% PAGE/8 M urea is shown.