Aaron J. Shatkin Professor UMDNJ Dept. of Molecular Genetics and Microbiology CABM. Room 106A Hoes Lane Piscataway. NJ 08854 (732) 235-5311 FAX - 5318 shatkin@cabm.rutgers.edu

Mechanisms of eukaryotic gene expression and viral and cellular transcriptional and translational processes

One of the earliest steps in the cascade of controls that regulate mRNAformation and function is the addition of a 5'-terminal "cap." This structural hallmark is present on all eukaryotic cellular mRNAs and is essential for viability. The cap enhances several downstream events in gene expression including splicing of pre-mRNAs in the nucleus. initiation of protein synthesis. and mRNA stability. These important effects have fostered many studies that have defined the enzymatic mechanisms of capping. Recently we have cloned and sequenced the mouse and human capping enzymes and mapped the human protein to 6q16. a region implicated in tumor suppression. The mammalian capping enzymes are 597-amino acid. 68kD polypeptides consisting of two functional domains-N-terminal RNA 5' triphosphatase (RT) and C-terminal guanylyltransferase (GT). Mutational analysis demonstrated that the GT active site lysine is present in the sequence 294 Lys-X-Asp-Gly 297. one of several highly conserved motifs characteristic of a nucleotidyltransferase superfamily of proteins that includes other cellular and viral capping enzymes. A haploid strain of S. cerevisiae lacking mRNA guanylyltransferase was complemented for growth by the mouse wild type cDNA clone but not by a clone containing alanine in place of lysine in the KXDG motif. The results demonstrate the functional conservation of capping enzymes from yeast to mammals and open new possibilities for defining early events in the activation of gene expression.

We found that mammalian capping enzyme binds via its GT region to the hyperphosphorylated C-terminal domain (CTD) of RNA polymerase II. explaining the selective capping of pre-mRNAs. Similarly. the full length and C-terminal fragment of capping enzyme. but not the N-terminal domain. were localized to the nucleus in transfected cells and also bound poly (U) in vitro. Thus. the C-terminal domain of capping enzyme accesses nascent transcript 5'termini directly by RNA binding. dependent at least in part on electrostatic interactions. The capping enzyme N-terminal RNA 5'-triphosphatase (amino acids 1-237) contains the sequence VHCTHGFNRTG which corresponds to the conserved active-site motif in protein tyrosine phosphatases (PTPs). Mutational analyses identified the Cys and Arg residues in this motif and an upstream aspartate as important for triphosphatase activity. These and other results indicate that removal of phosphate from RNA 5'ends and from modified tyrosine residues in proteins occurs by a similar mechanism.

We have also cloned and characterized the third essential enzyme for mRNA 5'-capping-human mRNA (guanine-7-) methyltransferase (MT). It was mapped to 18p11.22-p11.23. a region encoding brain transcripts that have been suggested as positional candidates for susceptibility to bipolar disorder. Sequence alignment of the 476-amino acid MT protein within the corresponding yeast. C. elegans and Drosophila enzymes demonstrated several required. conserved motifs including one for binding S-adenosylmethionine. MT bound to human capping enzyme and also formed ternary complexes with the hyperphosphorylated. elongating form of RNA polymerase II.

To identify other proteins that interact with capping enzymes. we used a yeast two-hybrid system to screen a human fetal brain cDNA library with full length human capping enzyme and isolated transcription elongation factor SPT5. It bound to capping enzyme and stimulated RNA guanylylation but not the triphosphatase step of capping. Purified. hyperphosphorylated CTD similarly stimulated RNA guanylylation. but the effects of P-CTD and SPT5 were not

additive,suggesting a common binding site on capping enzyme. By using two-hybrid. GST-pulldown and co-immunoprecipitation approaches. we also found that MT interacts with the nuclear transporter. importin-alpha (Impa). MT selectively bound and methylated RNA containing 5'-terminal GpppG. and both activities were stimulated several-fold by Impa. MT/RNA/Impa complexes were dissociated by addition of Imp-beta (Impb) which also blocked Impa stimulation

of RNA cap methylation. RanGTP but not RanGDP prevented these effects of Impb. The results suggest that. in addition to a linkage between capping and transcription. mRNA biogenesis and nucleocytoplasmic transport are functionally connected. a possibility which we are exploring.

Selected Publications

Das K, Bauman JD, Clark AD Jr, Frenkel YV, Lewi PJ, Shatkin AJ, Hughes SH, Arnold E. (2008)
High-resolution structures of HIV-1 reverse transcriptase/TMC278 complexes: strategic flexibility explains potency against resistance mutations. Proc Natl Acad Sci U S A. 105(5):1466-71.

Kaneko S, Chu C, Shatkin AJ, Manley JL. (2007) Human capping enzyme promotes formation of transcriptional R loops in vitro. Proc Natl Acad Sci U S A. 104(45):17620-5.

Shafer. B.. Chu. C.. and Shatkin. A.J.(2005). Human mRNA Cap Methyltransferase: Alternative nuclear localization signal motifs ensure nuclear localization required for viability. Mol. Cell. Biol. 25:2644-49.

Furuichi. Y. and Shatkin. A.J.(2004). mRNA Capping-Eukaryotic Hallmark. Encyclopedic Reference of Genomics and Proteomics in Molecular Medicine.

Mandal. S.S.. Chu. C.. Wada. T.. Handa. H.. Shatkin. A.J.. and Reinberg. D.(2004). Functional interactions of capping enzyme with factors that positively and negatively regulate transcription during promoter escape. Proc. Natl. Acad. Sci. USA 101:7572-77.

Srinivasan. P.. Piano. F.. and Shatkin. A.J.(2003). mRNA Capping enzyme requirement for C. elegans viability. JBC 278:14168-14173.

Zhou. M.. Deng. L.. Kashanchi. F.. Brady. J.N.. Shatkin. A.J.. and Kumar. A.(2003). The Tat/TAR-dependent phosphorylation of RNAP II CTD stimulates cotranscriptional capping of HIV-1 mRNA. Proc. Natl. Acad. Sci. USA 100:12666-71.

Shatkin. A.J.(2000). Reovirus Translational Control. In Translational Control. Sonenberg. N.. Hershey. J.W.B.. Mathews. M.B.. eds. Cold Spring Harbor Laboratory Press. 2nd edition. 915-932.

Furuichi. Y.. and Shatkin. A.J.(2000). Viral and cellular mRNA capping: Past and prospects. Adv. in Vir. Res. 55:135-184.

Shatkin. A.J.. and Manley. J.L. (2000). The ends of the affair: Capping and polyadenylation. Nature Structural Biology 7:838-842.

Wen. Y.. and Shatkin. A.J.(2000). Cap Methyltransferase selective binding and methylation of GpppG-RNA Are stimulated by Importin-. Genes and Development. 14:2944-2949.