AARON J. SHATKIN
Director
Center for Advanced Biotechnology and Medicine

Professor
Department of Molecular Genetics and Microbiology
UMDNJ – Robert Wood Johnson Medical School

University Professor of Molecular Biology
Rutgers, The State University of New Jersey

Member
Cancer Institute of New Jersey

Ph.D., 1961, The Rockefeller University

Telephone: (732) 235-5311
Fax: (732) 235-5318

mRNA processing, 5'-capping, cap methyltransferase, transcription

One of the earliest steps in the cascade of controls that regulate mRNA formation 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. We have cloned and sequenced the mouse and human capping enzymes and mapped the human protein to 6q16, a region implicated in tumor suppression. Both of the 597-amino acid, 68kd mammalian polypeptides consist 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 required 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-' (Imp' ). MT selectively bound and methylated RNA containing 5'-terminal GpppG, and both activities were stimulated several-fold by Impα . MT/RNA/Impα complexes were dissociated by addition of Impβ which also blocked Impα stimulation of RNA cap methylation. RanGTP but not RanGDP prevented these effects of Impβ . The results suggest that, in addition to a linkage between capping and transcription, mRNA biogenesis and nucleocytoplasmic transport are functionally connected.

Modulation of capping. a, Pol II A form containing unphosphorylated CTD initiates transcription, produces 20-25 nucleotide 5'-triphosphorylated transcripts and pauses with SPT5 bound as part of a large transcription complex. b, CTD is phosphorylated in the O form, changes conformation and binds capping enzyme (CE). CE modifies the exposed end of the nascent transcript, stimulated by SPT5 as well as P-CTD. MT binds to CE (mammals) or P-CTD (yeast) and to the terminal GpppN (mammals). c, Impα stimulates MT substrate binding and N7 methylation of the cap G, and Pol II O form switches from initiation to processive elongation.

Selected Publications

Topisirovic, I., Svitkin, Y., Sonenberg, N., and Shatkin, A.J. Cap and Cap-binding Proteins in the Control of Gene Expression. WIREs RNA, 2010 (in press).

Shatkin, A.J., Das, K., and Arnold E. 3D Jigsaw Puzzle in Rotavirus Assembly. Structure 16:1601-2, 2008.

Bauman, J.D., Das, K., Ho, W.C., Baweja, M., Himmel, D.M., Clark, A.D. Jr., Oren, D. A., Boyer, P. L., Hughes, S. H., Shatkin, A. J., and Arnold, E. Crystal engineering of HIV-1 Reverse Transcriptase for Structure-based Drug Design. Nucl. Acids Res. 36:5083-92, 2008.

Chu, C., and Shatkin, A.J.: Apoptosis and Autophagy Induction in Mammalian Cells by siRNA Knockdown of mRNA Capping Enzymes. Mol. Cell. Biol. 28:5829-36, 2008.

Liu, D., Fritz, D.T., Rogers, M.B., and Shatkin, AJ.: Species-specific Cis-regulatory Elements in the 3′UTR Direct Alternative Polyadenylation of BMP2 mRNA. J. Biol. Chem. 283:28010-9, 2008.

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

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

Shafer B, Chu C, Shatkin AJ. (2005) Human mRNA cap methyltransferase: alternative nuclearlocalization signal motifs ensure nuclear localization required for viability. Mol Cell Biol 25:2644-9

Mandal SS, Chu C, Wada T, Handa H, Shatkin AJ, Reinberg D. (2004) Functional interactions of RNA-capping enzyme with factors that positively and negatively regulate promoter escape by RNA polymerase II. Proc Natl Acad Sci USA 18:7572-7

Shatkin AJ, Manley JL. (2000) The ends of the affair: capping and polyadenylation. Nat Struct Biol 7:838-42

Yue Z, Maldonado E, Pillutla R, Cho H, Reinberg D, Shatkin AJ. (1997) Mammalian capping enzyme complements mutant Saccharomyces cerevisiae lacking mRNA guanylyltransferase and selectively binds the elongating form of RNA polymerase II. Proc Natl Acad Sci USA 94:12898-903