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Dr. Arthur D. Eisenberg Associate Reseazr.h Director The Council for Tobacco Research- USA, Inc. 900 Third Avenue New York, NY 10022 Dear Dr. Eisenbe~g: Encl~l please find a Preliminary Application requ¢ : "Intexnal Methylafion of mRNA in Normal and Tran~ . .. • form, ~ projec~ is not supported by any major fund. :, • - : .... ,__ .... ~ we have plans to request support. We have been cau~.~ u~-j~-cf il~r~u-g~i institutional support. However, I believe that our recent results bodes well for substantial progress in understanding the function of this ubiquitous mRNA modification and we now seek support to continue these studies. I anticipate thac the duration of the studies outlined in this preliminary proposal will encompass three years. Support at the level of $80,000 is requested for the first year of the study. Sincerely, Professor and Chairman (216) 368-3420

February l3, 1996 Dr. Amhur D. Eisenberg Associate Research Director The Council for Tobacco Research - USA, Inc. 900 Third Avenue New York, NY I0022 Dear Dr. Eis~nberg: Enclosed please find a Preliminary Appli~tion requesting research support for a project entitled, "Internal Methylation of mRNA in Normal and Transformed Cells." As indicat~l in the attached form, this project is not supported by any major funding sources including NIH, NSF or ACS, nor do we have plans to request support. We have been carrying this project through institutional support. However, I believe that our recent results bodes well for substantial progress in understanding the ftmction of this ubiquitous mRNA modification and we now seek support to continue these studies. I anticipate that tim duration of tim studios outlined in this preliminary proposal will encompass three years. Support at the level of $80,000 is requested for the first year of the study. Sincerely, F~~oottman, Ph.D. Professor and Chairman (216) 368-3420 FMP./ac

Desp~ eatensive efforts in a number of hboramri~, neitl~r th~ molecular ch~-acteri~tt~n of this enz'~ re~on nor the biologic~_l ftmc~n of mSA me~hyhlioa in ml~A l~ be~_n defined. ~ ~ ~ ~ ~y ~o~ ~ of ~A ~m~ ~ ~n ~~ ~y ~ ~ ~ p~ ~ ~ ~ m ob~ a ~NA ~ ~r ~ ~e. We ~ve ~n~ ~oI~ ~ a cl~ ~ ~ ~w a~ ~ d~ ~s ~~ ~ ~~ m~A ~on h ~A, im ~ biological ~fion ~ s~c~y, ~ role R ~t play h ~H~ Background Internal m6A residues are found in nk~NA sequences of all higher eukaryot~es examined, including plants, and also viruses whicl~ replicate within the nucleus. The level of m6A in a typical mammalian mRNA averages from three to five m6A residues ~r molecule. Early experiments suggested that meA o~curred exclusively within exons, but ree.enfly~m A has been found in intron-specific sequences of individual cellular mRNAs. The presence of m A in pre-mR~A introns and viral RNA sequences found in the nucleus is consistent with a nuclear role for this modification. In both heterogeneous nuclear RNA and cytoplasmic mRNA, only AAC and GAC sequences are methylated, where the underlined A residue denotes m6A. GAC and AAC sequences should appear every 32 nucleotides in a random sequence, yet only one or two m6A residues are found per approximately 1,000 nucleotides in mRNA. It is now known that methylation occurs within the extended consensus sequence_ NtRACN2, in which NI is a purine 90% of the time, and N2 is rarely guanine. Specific sites of m~'A residues have been identified in only two RNA sequences, the virion mRNA of RSV and prolactin (PRL) mRNA. Out of a total of 27 potential GAC and AAC methylation sequences present in PRL mRNA, a single AGACU sequence was methylated. Although experimental results to-date collectively support an extended consensus methylation site of RGACU, many such sequences are not utilized. This has been attributed to "context effects" in which strong positional effects or RNA secondary structure form part of the recognition site for methylation. The establishment of an in vitro cell-free system, utilizing HeLa cell nuclear extract and a segment of PRL mRNA as sub~strate, was used to define the substrate specificity and purification of the mRNA MTase. This in vitro methylation of a synthetic PRL mRNA sequence mimicked that found in vivo in the pituitary, occurring at the identical adenosine residue. Early fractionation steps appeared promising, but then resulted in a loss of enzymatic activity, initially ate"outed to enzyme instability. Only after complementation with various fractions of the partially purified MTase did we discover the multi- component character of this enzyme. We now know that three ~-l~.xate protein fractions, A1, A2 and B are required for enzymatic activity and this activity can only be recon~tuted by mixing theee three fractiom. A2 and B have been highly pur~.ed and in turn are made up of multiple subunits that diaso~iate upon SDS gel electruphore~is.

that m'A nny affect pre-mRNA splicing or mRNA tnmp~ from ~ to cTtoplamn. AIthough the biological role of internal mRNA methylation is poorly understood, several studies have noted a general alteration in the levels of RNA methylation in malignant ceils. Erythroleukcv3ic cells in culture have been used as a model sTstem to study the transition from~ a prolifvratPce, non-differea~ted cell to the differentiated state. Methylation inhibitors which block m6A methyladon in mRNA inhibit the transition of erythrol~c cells to the differentiated state. However, the lack of specificity of the methylation inhibitors ~used in these studies raises serious concerns regarding the correlation between disruption of mRNA m6A methylation and cellular proliferation or differentiation. Nonetheless, in preliminary studies we have measured m6A MTase activity in nuclear extracts from K562 cells before and afar induction to differentiation. Nuclear extracts prepared from cells triggered to undergo differentiation reproduc~ly demonstrated a three to ten fold decrease in m6A MTase activity relative to controls. The availability of a cloned eDNA sequence encoding an m6A MTase subunit, as described below, will allow us to directly address these questions. Specific Aims The availability of a eDNA clone for one of the critical MTase subunits now permits the molecular characterization of this complex enzyme. Furthermore, it allows us to directly assess the role of mRNA m6A methylation in post-transcriptional regulation, particularly as it relates to cell-cycle control, cellular transformation and induced carcinogenesis. These studies will be pro:sued under the following three specific aims: 1) Characterize, at a molecular level, the 70 kDa subunit of mRNA MTase A2 and verify its functional activity in binding SAM and complementing fractions A1 and B in in vitro methylation assays. 2) Utilize recombinant p70A2, as described below, to isolate and characterize additional components of the mRNA roSA MTase. 3) Employ pTOA2-specific antibodies and antisense sequences to explore the subcellular localization of this MTase and determine its regulated expression in normal and transformed cells. Recent P~ogress (Unpublished) Purification of the individual components of the m6A MTase provided an opportunity to attempt eDNA cloning using amino acid sequence data derived from purified subunit protein. Although our most purified preparations of fraction A2 are not homogeneous, a gel isolated subunit of A2 was identified functionally by SAM binding and its co-purification with m6A MTase activity. Using this approach, we obtained amino acid sequence for three peptides (6 to 15 amino acids long) from the homogeneous 70 kD SAM-binding subunit of A2. This amino acid sequence inform_~ion was used to generate three ~ts of degenerate oligmmcleotides which were then employed as probes to ~creen a eDNA library. We now have multiple, independeat 2.2 Kb eDNA ~ (pTOA2) ~ an open reading frame tlmt ~Jndm the precise aequence of all ~ pepfides. This eDNA ckme is literally the culmimtion of

TI~ ~ pT0.K2 cD~A ¢tmm will be exImmmd im a tmclm'iai ~ vect~ wi~ m~ mnino- compcmvnm A1 andB inthv/n v/tro m6A MTasv amay. Purifiedp70A2 will also beuscd to gencxatv a~tibodivs which in turn will be employed to block/n v/tro MTase activity of componem A2 in the reco~ ~msay. Additionally, these ~ will be used in subseqtmm evaluation of MTase levels and cellular localization studies as described below. Recombinant p70A2 will be used to identify and isolate remaining subunit proteins of the mRNA MTase hole-enzyme. First, recombinam p70A2 will be used to sequester specifically associated proteim that are part of the multi-component MTase. This will take advantage of the ability of the (Fits)s-tagged protein to be tightly bound to Ni-co ~baxms before and after component intex~.ction. Secondly, recombinant p70A2, labeled by p3Z kinasing of a small, fused pepfide sequence tag, will used to identify associated MTase subunits. This will be accomplished by far-we.stem affinity blotting of purified AI and B MTase component fractions. Studies of these associated A1 and B MTase components will include functional reconstitution, MTase assays and/n v/ire complementation studies. Long-term studies will focus on the RNA and protein binding domains of these associated proteins and their interaction with known components of the pre- mRNA splicing machinery and nuclear transport systems. Studies on the biological role of mRNA m6A MTas~ will include subcellular localization of this enzyme, using the MTasv-sp~cific antibodies described above. Alternatively, chimeric p70A2 protein, containing the green fluorescent protein tag, will be used in atmmpts to demrmine coincidence of the m~A MTase with "speckles" (RNA splicing) or "nuclear pores" (transport), for example. The levels of m6A MTase p70A2 mRNA will be determined in an erythropoiedc cell lines such as K562 or MEL cells, during stages of either rapid proliferation or following induction to the differentiated state. Similar studies will be carried out in synchronized coils to determine if m6A MTas¢ levels fluctuate as a funstion of the cell cycle. Summary Both the ubiquitous presence of m6A in mRNA and the inherent complexity of the enzymatic machinery involved in its formation argue for a biological function that has been preserved throughout metazoans. Possession of a eDNA clone encoding a unique SAM-binding component of this mRNA modifying enzyme now enables us to directly characterize this multi-component, active complex and the relationship of its expression to the t~ansformed state.

INSWruTION.~ID LOOATION B.A. 19~9 1963 1963-1966 RESEARCH AND/OR PROFESSIONAL EXPERIENCE: Concluding with present position, list, in chronologioal order, pre~iou= eraployment, e0qaedence, and ho~om. Key pemonnel inctude the pdndpai iave~ligator =rid any other individuals who pan'tcipmte in bSe scte~'~ifm development or e~cec~rtion of the project. Key persotmel typically w~l intrude all iadMduak= with doctoral or other professional degme=, but in some pm]ect~ will include individuals at the masters or baccalaureate level provided t~ey oontribute in a substantive way to the =olentitlc development or execution of the pmjecL Include present rnernbemhip o~ any F~deml Government public adviso~ committee. Ust, in chronological order, the title, all au~or=, and compkfm reference~ to all publication~ dudng the past three years and to representative earfmr publications pertinent to this application. DO NOT EXCEED TWO PAGES. Professional _Experimce: 1961-1963 1963-1966 1966-1970 1970-1974 1974-1975 1974-1981 1994 1981-prescat Prcdoctaral Fellow (USPtIS), Dcpatlm~t of Bioclmmistty, Univczxity of Michigan Pt~aloctoral Fellow CLISPHS) and American Cancer Society F~llowshil~s, with Dr. M.W. Niz~nberg at NIH Associate Professor of Biochemistry, Michigan Stat¢ University Visiting Professor of Biochemistry, University of Brifi~ Columbia School of Modicine Professor of Biochomistry, Michigan Stare Univc~ty Visiting Professor, Dcpartm~t of Chomistry and Biochemistry, Uniwrsity of Colorado Professor and Chairman of Molecular Biology and Microbiology, Case Western Reserve University Honors and Advisory. Committees: 1974 American Cancor Society, Scholar Award 1978-1981 Member of Biochemist~ Study Section, Chairman 1980-1981 1988-1992 American Cancer Society, National Scientific Advisory Committee-Personnel, Research B ~blicatiom (Past ttLrC¢ years) Shimba, S., Bokar, LA., Rottman, F. and Rcddy, R. Accurar~ and efficiont N~-ad~osi~ methylatlon in spliccosomal U6 small nuclear RNA by HoLa cell extract in vitro. NucL Adds. Re#. 23 (13):2421-2426 (1995"). D~lcscn, W.P., Sun, Q. and Rotmmn, F. Multiple Splicing Signals Control Alt¢rnativo Intron Rstcntion of Bovh~ Growth Hormone Pre-mRNA. J. Biol. Chem. Z70(10):5346-5352 (1995). Rottman, F.M., Bokar, J.A., Narayan, P., Sbambaagh, M.E. and Ludwiczak, R. N6-Adenosine methylation in mRNA: Substrate specificity and enzyme complexity. Bioch~e 76:1-6 (1994). Bokar, J.A., Rath-Shambaugh, M.E., I.,udwiczak, R., Narayan, P. and Rottman, F. Characterizafi¢m and paxtial put,cation of mRNA N6-edenesine methyltrans~ase from HeLa eel[ nuclei. J. Biol. Chem. 269:17697-17704. (1994). Narayan, P., Ludwicz~, Goodwin, E.C. and Rottman, F.M. Context effects on N6A-adenosine methylation sims in prolactin mRNA. N~leic Acids Res. 22:419-426 (1994). Dirkse=3, W.P., ~, R.K., Sun, Q. and Rottman, F.M. A purJae-rich e~on sequence e~hances alte, u~ive splicin8 of bovine growth hormone pre-mRNA. J. Biol. Chem. 269:6431-6436 (1994). Sun, Q., Mayeda, A., ~, R.K., KraJaer, A.R. and Rottman, F.M. Gene~ splicing factor SF2/ASF promotes alternative Sun, Q., I-Iatapmn, R.IL, a=d Roltman, F.M. /~ ~/=ro aa=ly.,,i= ef bovi~ gme~ horma~ lm~-mRNA altem~ve ~liciag: n~pbed far ~and aee~atelJ~Tadm~J=ke~ L efBieL Cban. :NlT:l~30-16a34 (19~). ~ Bleclu=m. :~0~:147.,-149 5O544O87

I~j~mvic, A., De~s, R. E., S~emm, L N., Roe~ F. M. A sp~ced leader is preaent ~ a subset ~ mRNAs ft~m ~e Imr~ ~~~~ A ~ ~ ~ ~ ~ ~~ ~. ~. N~. ~. ~i. ~A ~:~17- ~~~. ~. ~. ~ 9:1~1610, (198~. N~P.~F.M.~~~of~~~~~~~~~ ~A. S~ ~: 11~11~ (I~). D~, R.B., A~. ~, S.M. ~, A. P~j~, ~ F.M. ~. T~y ~ ~ ~ 81 ~ ~ ~ ~, ~y ~ ~'~ ~J~ ~i~. Mol. ~ C~. ~. ~47~755 0988). ~, D.D., S. H~, J. ~h~t-~, $.A. ~~ F.M. ~ ~ K.R. Y~. H~~ ~ ~ ~: A ~ ~~ m~e ~t ~ ~ ~ ~ ~. ~ ~ ~I. 2:11~11~ D~, A.H., R. BI~, F. ~, ~ ~ ~ A. M~. ~ ~ eDNA c~ ~r ~~y ~ ~ of ~ h~ p~i~ Sc~ ~. ~. Natl. ~ Sd. U~ ~:553~5538 (198~. ~w~, R.G., F.M. ~ T. C~, H.J. K~, P.A. ~ ~ T.W. ~. c~B ~fivafi~ ~ a~ ~is ~-~ ~b~: ~e ~ ~ f~ ~r ~ ~ ~ by ~five ~A ~ss~. Mol. ~ ~. Biol. 6:31~-3133 (1986). D~, R.C., J. ~, ~ ~r, D.S~va, R.P. W~, D.D. S~ ~ F.M. ~. ~is of ~ ~ ~ ~ ~ by ~ a ~~ ~. ~1. ~ ~H. Biol. 5:~2~3 (19~. N~a~n, T.W., P.A. ~, R.G. ~, F.M. ~ L.B. ~R~ M.B. R~ ~ H.L K~. c~rb ~fivafi~ ~ ~V-~ ~~: ~ ~A ~s~ ~ p~ ~c~n r~t ~ ~n of ~ ~~at~ ~F ~. C~H 41:71~7~ (1~. Cm~r, S.A., Y.A.S. Y~ ~ F.M. ~. H~ ~ of ~ ~nc pm~ pm~ ~ rat ~ ~ ~. J. B~I. ~m. ~:1~1~1 (1~). Woy~, R.P., R.H. Lyom, L. ~st ~ F.M. ~. ~m~ ~r ~ 3" fl~ m~on of ~ ~ ~ ho~ g~ ~r ~a~ ~ly~yl~on. ~. Nail. Ac~. ~i. USA 81:3~3~8 (1~). N~mn, J.H., P.A. F~, J.B. V~, M.T. ~rb~, S.~ C~r ~ F.M. R~. ~vel~ exxon ~ ~ ho~o~ ~ ~ ~s ~ ~ ~ pi~. J. Biol. C~. ~8:4565 (1~3). W~, R.P., S.A. Cm~r, R.H. Lyom, S. H~w~, E.C. ~w~ ~ F.M. R~. Clo~ ~ n~fi~ ~~ of ~ ~ ~ ~ ~. N~c Aci~ ~ 10:7~ (19~). S~, N.L., J.H. N~ S. Holm ~d F.M. ~. N~I~ s~u~ of ~ prolac~ mesm~er ~A. J. Biol. C~. ~:678 (19~). S~v~e, N.L, M. ~ S. ~, C. ~11, J.H. Nil~ ~d F.M. Ro~. Us~ of ol~~leo~ pr~e~ ~ ~ ~(A)-Mj~t ~ ~ ~A: ~s 3"-~ n~ ~q~e of ~v~ ~ hom~ ~A. B~~ 19:1737 (19~). N~, J.H., K.$. ~er, E.M. C~, K. Ffi~i ~ F.M. ~. On~ of pi~ ~e ~ ~ ~ ~ fe~: ~~ of p~ml~n ~A ~ a ~ of g~fion. J. Bi~. ~m. ~:5~1 (19~). N~, J.H., E.M. ~n~ ~ F.M. ~m ~fi~fi~ ~ ~~ ~A ~m ~ ~ ~ ~. J. B~I. C~. ~:1516 (1~. ~, R.P., D.E. ~ly, K.H. F~i ~ F.M. ~. Me~y~ ~mfi~ of he~ge~ ~ ~A: pr~ ~bl~ 5"-~ a~. ~ 6:13 (1~. ~, ~E, D.E. ~, K. F~ ~ F. ~. ~ ~~ ~~ ~ ~H ~s~r ~A: ~ ~r ~ ~ ~m~ ~ ~ 5"-~. ~ 4:3~ (1~. ~, F., A~. ~~R.P. ~. ~ ~ ~y~~fi~ ~ 5"~ of~~: ~ ~~ ~ ~. ~ a:~ 0~4). D

INS'TTTi~ION AND LO~TION University of Cincinnati B.S. 1987 FIELD OF STUDY Biology RESEARCH AND PROFESSIONAL EXPERIENCE: Concluding w~h preeont position, list, in cl~ronological order, previous employment, experience, and honors. Include present membership on any Federal Government public advisory committee. List, in chronological order, the titles, all authors, and complete references to all pubEcations during the past three years and to representative earlier publications pertinent to this application. If the ist of publications in the last three years exceeds two pages, select the most pertinent publications. DO NOT EXCEED TWO PAGES. PROFESSIONAL EXPERIENCE: 198%1988 1988-1990 1990-1996 Research Assistant IL Dr. Jerry Lingrel, Molecular Biology, University of Cincinnati Research Assistant, Molecular Genetics Laboratory, Good Samaritan Hospital, Cincinnati, OH Research Assistant HI with Dr. Fritz M. Rottman, Case We.stem Reserve University REPRESENTATIVE PUBLICATIONS: Rottman, Bokar, Narayan, Shambaugh and Ludwiczak. NLAdenosine Methylation in mRNA: Substrate Specificity and Enzyme Complexity. Biochimie 76:1-6 (1994). Bokar, L, Shambaugh, M.E., Narayan, P. and Rottman, F.M. Characterization and partial purification ofmRNA N- adenosine methyltransferase from HeLa cell nuclei. J. Biol. Chem. 269:17697-17704 (1994). 50( ¢4089

Title ~fProject Post-transcriptional regulation of pituitary gene expression Post-transcriptional regulation of human growth hormone receptor in the placenta (Postdoctoral fellowship) Graduate Training in Cell and Molecular Biology (Training Grant) NIH, R01 DK32770- 11 NIH, F32 DK09420 NIH, 5 T32 GM08056- 13 $2,158,402 $67,250 $1,972,881 Cummt Almml Ammmt Av~l~bl= to You $192,111 $23,700 $189,653 7/31 ~87 3/31/98 6/30/97 Identify and describe any overlap of this application with the above grants: None Indicate the total annual funds availabl¢ to you this year for all research projects under your supervision. $192,111 PENDING OR PLANNED Title of Project None Planned: B. Internal Methylation of mRNA in Normal and Transformed Cells (this grant) Sout~¢s (give grant numbers) Tobacco Research Council Toted Value of Grant (direct costs) $240,000 Avg. Annual Amount Available to You $80,000 Total Duration (give inclusive dates) 1/1/97 - 12/31/99 and de~oeaibc any overlap of this applica~tion with the above project.

N~cl~ic Ack~s i~sean:~, I~5. VoL 2~ Ho. 13 2421-~42~ Accurate and efficient N-6-adenosine methylation in spliceosomal U6 small nuclear RNA by HeLa cell extract in vibo Shigeki Shimba, Joseph A. Bokar1, Fritz Rottman1 and Ram Reddy* Department of Pharmacology, Baylor College of Medmine, Houston, TX 77030, USA and 1Dep~rtment of Molecular Biology and Microbiology, Case Westem Reserve University, Cleveland, OH, USA Reoeived April 10, 1995; Revised and Aocepled May 31, 1995 ABSTRACT Human US small nuclear RNA (US snRNA), an abun- dant snRNA required for splicing of pre-mRNAs, contains several post-transcriptional modificalfons including a single roSA (N-6-methyladenosine) at peai- tion 43. This A-43 residue is cdtical for the function of U6 snRNA in splicing of pre-mRNAs. Yeast and plant U6 snRNAs also contain m6A in the corresponding position showing that this modification is evolutionadly conserved. In this study, we showthat upon incubation of an unmodified U6 RNA with HeLa cell extract, A-43 residue in human U6 snRNA was rapidly converted to roSA-43. This conversion was detectable as eady as 3 rain after incubation and was nearly complete in 60 rain; no other A residue in U6 snRNA was converted to roSA. Deletion studies showed that the stem-loop structure near the 5" end of U6 snRNA is dispensable for roSA formation; however, the integrity of the 3' steda-loop was necessary for efficient mSA formation. These data show that a short stretch of pdmary sequence flanking the methylation site is not sufficient for U6 mSA methyltransferase recognition and the enzyme probably recognizes secondary and/or tertiary structural features in U6 snRNA. The enzyme that catalyzes roSA formation in U6 snRNA appears to be distinct from the prolactin mRNA methyltransferase which is also present in HeLa nuclear extracts. INTRODUCTION U6 snRNA, an essential component for splicing of pre-mRNAs (1-3), contains several post-transcriptional modificati(ats (Fig. I). These include formation of y.monomcthyl phosphate cap sm~ctu~¢ (4~5), LTMP addkion on the 3' end (6-8), formation of a 2,3' cyclic phosphate at the 3" end (9), base mcthyladons including a N-6-methyladenosinc (m6A) formation at position 43 and ~¢vm'al 2"-O-ribose med~lafioa~ (10,11). The NJ:.,-m~yla,- doaof~e at posido~ A-43 of lamina U6 saRNA matur~ mRNAs, rRNAs, tRNAs, U2 snRNA and U4B snRNA. (ii) The position 43 in mammalian U6 snRNA is in a highly cor~"crved region (3). (iii) The mutation otithis nucieotide is lethal in yeast (12). (iv) Furthermore, cross-linking studies showed that this region of U6 snRNA encompassing mOA-43 is involved in beso-pairing with pre-mRNA (13-15). Recent data showed that A-43 of U6 snRNA is actually involved in bass-pairing with the prc-mRNA sexjucncc near the 5" splice-site ( 16,17). These studies show that m~A in U6 snRNA is present in a functionally important nuclcotidc; however, the function of roSA residues in snRNAs, raRNAs or in other RNAs is not known. The synthesis of roSA in mRNAs has been studied by several investigators. The rnSA in mRNAs has been found in a variety of organisms including higher eukaryotes and in viral RNAs (reviewed in 18). The number of mSA r~sidu~s varies among different messenger RNAs. In HeLa, Novikoff and L-cells, the average mRNA molecule contains three mSA residues. Rous sarcoma and influenza viruses have 1-12 m~A sesidues per genomic RNA subunit (rcviewe~l in 18). The methyla.tion of N-6-adeninc in raRNAs occurs prior to their polyadenylation, and the roSA residues am conserved during processing and are present in mature mRNAs (19). In all known cases, the mSA residues are found in two conserved sequences, AmSAC or GmSAC (20-22). Cespany et el. (23) have confn'racd and extended the comus sequence for rncthylation to RGraSACU, whom R is usually a guanine residue. In addition to the primary sequence, efficiency of roSA mcthyladon in mRNAis influenced by the ovexall context in which the consensus sequence is located (24). However, the primary sequence around the methylation site A-43 in human U6 snRNA is UACm6AGA showing that the methylation site in U6 snRNA does not match the consensus sequence found in ml~. As. To understand the mechanism of this faithful and sequence- dependent methyl transfer reaction which accurately methylates the/¢-6-posidon of adenine residues in mRNA, a odl-free syslem wa~ developed (25). Using this in vitr~ systera, the factors involved in m6A formation in taR.HAs have 1xen partially pmif~ed from HeLa ce~ (26,2"/)..While dgaifc~f in'ogress has been made in clm'acte~ag the meA formation in mRNA~ not Imm~ U6 saRNA miag aa ~ ~ro ~. "Pan ~lata laeaented 50544O91