Transcription elongation factor hSPT5 stimulates mRNA capping

Abstract

RNA polymerase II nascent transcripts are capped during pausing before elongation. Here we report that hSPT5, the human homolog of yeast elongation factor SPT5, interacts directly with the capping enzyme. hSPT5 stimulated capping enzyme guanylylation and mRNA capping by severalfold. Although RNA 5'-triphosphatase activity was unaffected, binding to this domain in the full-length enzyme is likely involved in the stimulation, as hSPT5 did not increase the activity of the guanylyltransferase fragment. Consistent with capping enzyme binding, TFIIH-phosphorylated CTD stimulated guanylylation, and this increase was not additive with hSPT5.

Figure 1

Mammalian capping enzymes interact with hSPT5 in vitro and in vivo. (A) Binding to HCE. Purified GST–hSPT5 was incubated with purified HCE (lane 1). Bound HCE was detected by SDS-PAGE followed by immunobloting with anti-MCE antisera (Yue et al. 1997; lanes 2,3) and densitometry. (Input lane) 50% of the amount of HCE used for the binding assays. (B) Schematic diagram of the MCE, RTP, and RGT domains. (C) Purified GST, GST–RTP, GST–RTP (C126S), GST–RGT, or GST–RGT (K294A) was incubated with [³⁵S]Met-labeled hSPT5. Bound proteins were separated by SDS-PAGE followed by autoradiography. (Input lane) 10% of the amount of hSPT5 used for the assays. (D) Schematic diagram of hSPT5 protein constructs used to define sequence requirements for interaction with HCE. (E) Full-length hSPT5 and indicated truncation mutants were transformed into yeast Y190 harboring HCE. Yeast colonies that grew on SD/−Trp/−Leu were assayed for liquid β-gal activity.

Figure 2

hSPT5 increases RGT and capping but not RTP activity. HCE (3.5 pmoles) was assayed for RTP activity (A), guanylylation (B), and RNA capping (C) in the presence of increasing amounts of hSPT5 (0, 7, 14, 28 pmoles) as described in Materials and Methods. (A) ³²Pi released from γ-³²P-labeled runoff transcripts was detected by TLC. Guanylylated HCE (B) and capped RNA (C) were analyzed by PAGE and autoradiography.

Figure 3

RTP–hSPT5 interactions can reverse guanylylation stimulation. (A) Purified RGT fragment (3.5 pmoles) was incubated with [α-³²P]GTP in the presence of 0, 7, 14, and 28 pmoles of hSPT5 and assayed as in Fig. 2B. (B) HCE (3.5 pmoles, lanes 1–5) was assayed for guanylyltransferase activity in the presence of hSPT5 (7 pmoles, lanes 2–5) and 7, 14, or 28 pmoles of MCE–RTP fragment (lanes 3–5). Guanylylation of ³²P-labeled HCE was quantitated by PhosphorImager after SDS-PAGE.

Figure 4

Stimulation of capping reaction by P-CTD is not enhanced by hSPT5. (A) CTD was phosphorylated by incubation with TFIIH, and the effect of 0, 7, 14, or 28 pmoles on HCE guanylylation was assayed as in Fig. 2B. (B) TFIIH-phosphorylated CTD was incubated with HCE (3.5 pmoles), [α-³²P]GTP and runoff transcripts and assayed for RNA capping as in Fig. 2C. (C) hSPT5, P-CTD or both proteins (0, 7, 14, 28, 56 pmoles) was incubated with 3.5 pmoles of HCE and [α-³²P]GTP and assayed for guanylylation as in Fig. 2.