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Biology Articles » Molecular Biology » RNA polymerase II and the integration of nuclear events » mRNA capping

mRNA capping
- RNA polymerase II and the integration of nuclear events

It has been known for some time that the cap structure found at the 5' end of all eukaryotic mRNAs is formed shortly after transcription initiation, when nascent RNA chains are about 25-30 nucleotides in lengths (see, for example, Coppola et al. 1983; Jove and Manley 1984). Capping is carried out by a series of three enzymatic activities (for review, see Shuman 1995). RNA triphosphatase removes the gamma-phosphate of the first nucleotide of the pre-mRNA, followed by the transfer of GMP to the resulting diphosphate end by RNA guanylyltransferase. RNA (guanine-7-) methyltransferase then adds a methyl group to the N7 position of the cap guanine to form the m7G(5')ppp(5')N cap. In metazoans, the capping enzyme is bifunctional with both RNA 5-triphosphatase and RNA guanylyltransferase activities, while in Saccharomyces cerevisiae, capping enzyme consists of a heterodimer of RNA triphosphatase (Cet1) and RNA guanylyltransferase (Ceg1). Although capping has been known to occur only on RNAs made by RNAP II, the mechanism for this specificity and what insures rapid, efficient capping has been elucidated only recently.

We now know that RNAP II0, and specifically the CTD, plays a direct role in the capping reaction (Fig. 2). Mammalian capping enzyme can directly and selectively bind RNAP IIO (Yue et al. 1997) and the phosphorylated form of a recombinant glutathione-S transferase-CTD fusion protein (GST-CTD; McCracken et al. 1997a; Ho et al. 1998a) through its guanylyltransferase domain. In budding yeast, guanylyltransferase activity can be recruited by phosphorylated CTD, but not by unphosphorylated CTD, from a purified capping enzyme preparation (Cho et al. 1997), and recombinant Ceg1 and methyltransferase (Abd1) directly and independently bind to the phosphorylated CTD (McCracken et al. 1997a). Supporting the functional significance of these interactions, RNAs transcribed by RNAP II with a shortened CTD undergo inefficient capping in transiently transfected mammalian cultured cells (McCracken et al. 1997a). In addition, a viable truncation mutant of the yeast CTD was found to be synthetically lethal in combination with a capping enzyme mutant (Cho et al. 1997). Thus, these phosphorylation-dependent interactions between the capping apparatus and the CTD are evolutionarily conserved and likely provide a basis for the specific and rapid targeting of the capping enzyme to RNAP II transcripts.

Important extensions of the recruiting model of capping enzyme by the phosphorylated CTD have been made in the last two years. In the first step of the guanylyltransferase reaction, the enzyme itself is guanylylated to form a covalent enzyme-GMP intermediate. Despite the fact that Ceg1 can bind to phosphorylated GST-CTD (McCracken et al. 1997a), guanylylation activity, as measured by formation of the enzyme-GMP intermediate, could not be detected associated with GST-CTD (Cho et al. 1997). These seemingly conflicting findings were explained in an interesting way by Cho et al. (1998), who found first that phosphorylated, but not unphosphorylated, GST-CTD can actually inhibit Ceg1 activity, and second that the inhibition could be reversed and guanylylation activity actually enhanced, by addition of the Cet1 triphosphatase (Fig. 2). These findings and others suggest that Ceg1 activity can be allosterically regulated by interaction with both the Cet1 triphosphatase (see also Ho et al. 1998b) and RNAP II0. The authors suggest that the observed inhibition of guanylyltransferase by CTD is designed to prevent spurious enzyme activity and to coordinate guanylylation with triphosphatase activity.

Mammalian capping enzyme activity is also allosterically regulated by interaction with the phosphorylated CTD (Ho and Shuman 1999). The isolated carboxy-terminal guanylyltransferase domain of mouse capping enzyme was shown to bind synthetic CTD peptides containing phosphoserine at either position 2 or 5 of the heptad YSPTSPS repeat, but not to unphosphorylated CTD peptides. The CTD phosphopeptides containing phosphoserine at position 5 stimulated formation of the enzyme-GMP intermediate by enhancing the enzyme's affinity for GTP. However, CTD peptides containing Ser-2-phosphorylation had no effect, either on basic enzyme guanylylation or on guanylylation activated by the Ser-5-phosphorylated peptide, indicating that the guanylyltransferase domain of the mammalian enzyme has two independent binding sites for the phosphorylated CTD: one is specific for the Ser-2-phosphorylated peptide and the other is an allosteric activator site recognized by the Ser-5-phosphorylated peptide (Fig. 2; Ho and Shuman 1999). Why in yeast Ceg1 is inhibited by phosphorylated CTD (in the absence of Cet1), whereas the mammalian guanylyltransferase domain is activated is unclear, but it may reflect the fact that transferase and triphosphatase activities are contained in the same polypeptide in mammals but not yeast. Thus the need to coordinate guanylyltransferase and triphosphatase activities does not exist in mammals. Together, these findings have suggested that the phosphorylated CTD functions not only as a simple landing pad for capping enzyme but also as an important regulator of enzyme activity, with activation correlated with position-specific phosphorylation (serine 5) within the CTD heptapeptide.

Genetics studies have revealed that a specific CTD-kinase, Kin28, is likely required for recruiting capping enzyme to RNAP II in yeast (Rodriguez et al. 2000). Three kinases, which have all been implicated in phosphorylation of the CTD in S. cerevisiae, were tested for their ability to allow recruitment of capping enzyme to the CTD. These included the Kin28-Ccl1 complex, a component of general transcription initiation factor TFIIH (Svejstrup et al. 1996); the Srb10-Srb11 complex, which is associated with RNAP II holoenzyme (Liao et al. 1995), and CTDK-I (Sterner et al. 1995). Combinations of mutant alleles of the genes encoding these kinases were tested with a ceg1 temperature-sensitive (ts) mutant, which previously had been shown to exhibit synthetic lethality with a viable CTD truncation mutant (Cho et al. 1997). Although all of the kinases were able to phosphorylate GST-CTD to allow recruitment of capping enzyme in vitro, only kin28 mutant alleles exhibited a genetic interaction with the ceg1 mutant. The level of CTD phosphorylation and, intriguingly, Ceg1 protein levels were reduced in both the CTD truncation mutant and kin28 mutants, raising the possibility that Ceg1 associated with CTD phosphorylated by Kin28 may be stabilized relative to unbound Ceg1. Furthermore, conditional mutants in which serine 5, but not serine 2, residues were replaced with alanines in either the first or second half of the CTD were synthetically lethal in combination with a ceg1 mutant. These data are in good agreement with the CTD phosphorylation requirements of mammalian capping enzyme (Ho and Shuman 1999), and strongly suggest that TFIIH-associated Kin28 phosphorylates serine 5 of the CTD repeat, at least in part, to target and activate the capping apparatus.

An interesting extension to these findings was suggested by the discovery that a protein implicated in transcriptional elongation, hSPT5, interacts physically and functionally with human capping enzyme (Fig. 2; Wen and Shatkin 1999). SPT5 was initially uncovered genetically in S. cerevisiae (Swanson and Winston 1992), and functions together with SPT4 to modulate an early step in transcription elongation in both yeast and humans (Hartzog et al. 1998; Wada et al. 1998a). hSPT5 was isolated in a yeast two-hybrid screen with human capping enzyme as bait, and the two proteins were shown to interact directly in vitro. hSPT5 strongly stimulates the guanylyltransferase but not the RNA triphosphatase activity. Intriguingly, no stimulation of capping was detected when hSPT5 was added together with a phosphorylated GST-CTD protein, raising the possibility that the two capping activators function redundantly. Given that Spt5 interacts preferentially with RNAP IIA (Wada et al. 1998b), two models can be suggested to explain the role of hSPT5 in capping. In one, suggested by the authors, SPT4/5 dissociates from the CTD upon phosphorylation, but remains associated with the transcription complex and functions to enhance capping upon recruitment of capping enzyme to the phosphorylated CTD. In a second model, hSPT5 could function to recruit capping enzyme to the holoenzyme in some cases prior to, or independent of, CTD phosphorylation. Capping enzyme could then be transferred to the CTD upon phosphorylation and transcription, or be activated by hSPT5 to ensure rapid and efficient capping of transcripts that may be initiated and elongated prior to, or in the absence of, CTD phosphorylation. RNAP IIA has in fact been implicated in the elongation of a small number of genes (Weeks et al. 1993).

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