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[OLECULAR AND CELLULAR BIOLOGY A publication o~ the Arnerfcan Socfety [or MicroMo~ogy Manuscript Number:. MCBI445.92 Title: "A unique ribonucleoprotein complex..." 21 December 1992 Dr. Sally A. Amero Dept. of Molecular and Cellular Biochemistry Strich School of Medicine Loyola University Medical Center 2160 S. First Avenue Maywood, IL 60153 FAX (708) 216-8523 Dear Dr. Amero, All three referees saw much that was good in this manuscript, but felt that the data for HRB/PEP interactions should be clearer, and the basis for differential sedimentation of hnRNP and PEP- containing complexes should be further investigated. Under the circumstances I would like to strongly encourage you to resubmit an expanded manuscript addressing the major comments of all three referees. Three complete copies of the revised manuscript would have to be sent directly to the ASM, together with a detailed covering letter stating that this is a resubmission of MCB 1445/92 and indicating your response to each comment of each referee. The ASM would then automatically forward the resubmitted manuscript and cover letter to me, and I would forward the new manuscript to the same referees for scrutiny. Sincerely, Alan M. Weiner Editor in Chief cc: ASM Publications 40000010

A Unique Ribonucleoprotein Complex Assemble~ Pr_e_f_erentially on Ecdysone-Responsive Sites in Dros~i~ite Running Title: The PEP Ribonucleoprotein Complex in Drosophila Sally A. Amero1", Joel W. Hockensmith2, Gopa Raychaudhuri3'4, and Ann L. Beyer4 1"Department of Molecular and Cellular Biochemistry, Loyola University Chicago, Stritch School of Medicine, Maywood, IL 60153. Corresponding author. 2Department of Biochemistry, University of Virginia School of Medicine, Charlottesville, VA 22908. 3Present address: Hepatitis Section, Laboratory of Infectious l~iseases, NIAID, National Institutes of Health, Bethesda, MD 20892. 4Department of Microbiology, University of Virginia School of Medicine, Charlottesville, VA 22908. "TEL: 708-216-3365 FAX: 708-216-8523 40000011

ABSTRACT The Protein on Ecdysone Puffs {PEP) is associated preferentially with active ecdysone-inducible puffs on Drosophila polytene chromosomes and contains motifs characteristic of transcription factors and RNA-binding proteins (Amero, S.A., Elgin, S.C.R., and Beyer, A.L. Genes Dev. 5:188-200, 1991}. Using cytological and biochemical approaches, PEP was found to be integral to a chromosomal ribonucleopr'otein (RNP) complex. Its distribution on polytene chromosomes was similar to that of the HRB proteins - which are basic Drosophila hnRNP proteins (Raychaudhuri, G., Haynes, S.R., and Beyer, A. L. Mol. Cell. Biol. 12:8z~7-855, 1992) - with PEP sites comprising a large subset of HRB protein sites. In sucrose density gradients the PEP RNP complex is large, reasonably abundant, and nondiscrete; it sediments differently than the RNP complex containing the HRB proteins, suggesting that the PEP and HRB RNP complexes can exist independently. Possible associations between the complexes were revealed by the highly-specific retention of portions of PEP and of all the major HRB isoforms on an anti-PEP antibody column; RNAse digestion released a specific subset of HRB polypeptides. These observations lead us to suggest that a PEP/RNA complex assembles preferentially on ecdysone-regulated genes in Drosophila, presumably to expedite the transcription and/or processing of these transcripts. Once assembled, PEP and HRB proteins may interact via both protein-protein and protein-RNA interactions. 40000012

Independent deposition of heterogeneous nuclear ribonucleoproteins and small nuclear ribonucleoprotein particles at sites of transcription (mRNA si~l~˘ing/l~NA procc~SngO SALLY A. AMERO=˘, GOPA ~YCHAUDHU~*, CYNTHIA L. Cass*. ~ALTHER J. VAN VENROOU~, WINAND J. H~ETS~, ADRIAN R. K~INER[], AND A~ L. BEYER='** "~a~mem of Microblolo~. Onivc~ity of Vi~nh ~h~l o~ Medicine, ~aflottvsv~le. VA N~m~a. ~e Neth~rla~fls: and ~Cold S~ng H~r ~mtv~,'CoM S~ng H~r. NY 11~4 Communicated by Oscar L. Miller. dr.. dune 1~, 1~2 (received for re~ April IS. ABSTRACT The major nuclear ribonucleoproteins (RNPs) involved in prt-mRNA proccsslng are classified in broad terms either as small nuclear RNPs (snRNTs)~ which art major participants in the splicing reaction, or heterogeneous nuclear RNP$ (hnRNPs), which traditionally have been thought to function in general pre-mRNA packaging. We obtained antibodies that recognize these two clasr, es of RNP in Drosophila melanoga~ter. Using a sequential immunostaining technique to compare directly the distribution of tht, e RNPs on Drosophila polytene chromosomes, we found that the two patterns were very similar qualitatively but not quantitatively, arguing for the independent deposition of the two RNP types snd supporting a role for hnRNT proteins, but not snRNPs, in general transcript packaging. Both heterogeneous nuclear ribonucleoproteins (hnRNPs; reviewed in refs. 1 and 2) and small nuclear ribonueleopro- reins (snRNPs; reviewed in ref. 3) are deposited ˘otranscrip- tionally on eukaryotie RNA polymerase II transcripts (4-8). Whereas the major basic hnRNP proteins have been consid- ered traditionally to function in general pre-mRNA packaging (2, 9). they have been proposed recently to be specific splicing cofactors or to be preferentially associated with splice junction sequences (10-15). snRNPs are major partic- ipants in the splicing reaction (3) but have been implicated recently in general packaging as part of a previously assem- bled unitary processing complex also containing hnRNPs (5, 6). The various proposals predict different amounts and ratios of the two protein types on nuclear pre-mRNA molecules at chromosomal sites of transcription, which is the issue we have addressed by sequential immunostaining. The core hnRNP proteins fA, B, and C proteins of 32-45 kDa) were originally identified as the major proteins that are associated with newly synthesized pre-mRNA (in the form of 30-50S RNP particles) when it is extracted from nuclei fr~viewed in refs. I and 2). This observation, together with their nuclear abundance, their ability to bind single-stranded nucleic acids regardless of sequence, and their helix- destabilizing properties, led to the notion that these core hnRNP proteins are involved in general pre-mRNA packag- ing. much as the histones are involved in the general pack- aging of DNA (1, 2). However, more recent investigations of hnRNP proteins, using in vitro splicing or in vitro RNA binding studies, have suggested that these proteins play a role in the splicing reaction (10-12). that they bind with high affinity to sequences at 3" splice sites (13.14). and that they are dependent on snRNPs for acquisition of a erosslinkable association with RNA (13). These in vitro studies have led to TE.." ~ u~ !." ~-'atim~ costs of this art2d˘ were d,ffrayc"d in part by rage charge. l:a)'~,eaL T/~is article must th~rtt'vre be hereby marked "'~cI~ertisement'" ia zzeerda.,'.e˘ ',,˘ith 18 U.S.C. §1734 sc!-.ly to iEd'.˘ate this fuel a reappraisal of the independent structural role of hnRNP proteins in pre-mRNA packaging towards a view that they are a few of the many required cofactors for splielng. The simplest version of this view would predict a donstant stoi- chiometry of snRNPs and the core hnRNP proteins on pre-mRNA, in amounts that correlate with the number of splicing signals. Another recently proposed model would also predict a constant stoichiometry of snRNPs and hnRNP proteins on pre-mRNA, but in amounts that correlate with RNA length rather than with splicing signals (5, 6). The unitary processing complex proposal (5, 6) predicts codcposition and constant stoichiometry of hnRNP proteins and snRNPs on transcripts and is based on cytological observations ofoo~yte contents of the newt Notophthalmus viridescens. First, hnRNP pro- teins and snRNPs (plus other splicing factors) occur in the same nuclear extrachromosomai complexes, the B snurpo- sprees, and second, these same components bccur on almost all lampbrush chromosome loops in amounts that correlate with RNA mass distribution on that loop (5). Although reports of snRNPs at loci thought not to have introns [Chi- ronoraus polyten~chromosome Balbiani rings (4) and newt histone gone-containing lampbrash loops (5)] appear to sup- port this model, it is now known that the Balbiani ring genes contain introns (16, 17), and splicing signals may occur on extremely long (hundreds of kilobases) readthrough tran- scription units on lampbrush chromosomes (18). The original views that the major hnRNP proteins associate promiscuously with pre-mRNA, while snRNPs arc deposited specifically at splice sites, are supported by many in vitro RNA binding studies fe.g., refs. 8 and 19; reviewed in refs. 1-3), by analysis of KNA sequences associated with trotted hnRNP complexes (reviewed in ref. 1), and by electron microscopic visualization of active genes (20, 21). Thus the abundance of snRNPs at a given transcriptionally active site would reflect the number of introns and the strength of their splicing signals, whereas the abundance of hnRNP proteins would be a function of RNA length, leading to site-specific ratios of hnRNP proteins to snRNPs. Our observations of such site-specific ratios and of intense hnRNP staining at the highly transcribed puff sites support these original predictions. Abbreviations: hnRNP, heterogeneous nuclear ribonucleoprotein; snRNP, sma~! nuclear ribonu˘leol~mtein. *Present a~dress: Department of Molecular and Cellular Bioeh,~m- istry. Loyola University Medical Center, Maywood, IL 60153. Present address: Laborztor/of Molecular Genetics. National In- stitute of Child H,.,~Ith and Human Oevelol~meat. National Insti- tutes of Health. B,'thesda. MD 2~$92. "lpresent a~dress: Org2,r, on Teknika B.V.. l~oselr.d 15. PO Box 84. 5280 AB Boxtel. The Ne~erlands. °~I'o wh~rn reprint rtquests skv~Id be a~drcssed. 40000013

MINIREVIEW The Origin of Nuclear Receptor Proteins: A Single Precursor Distinct from Other Transcription Factors Sally A. Amero*, Robed H. Kretsinger, Nancy D. Moncrief, Keith R. Yamamoto, and William R. Pearson Department of Microbiology (S.A.A.) University of Virginia School of Medicine Charlottesville, Virginia 22908 Department of Biology (R.H.K.) University of Virginia Charlottesville, Virginia 22901 Virginia Museum of Natural History (N.D.M.) Martinsville, Virginia 24112 Department of Biochemistry and Biophysics (K.R.Y.) University of California at San Francisco San Francisco, California 94143-0448 Department of Biochemistry (W.R;P.) University of Virginia School of Medicine Charlottesville, Virginia 22908 Nuclear receptor proteins regulate transcription under the influence of hormones or other small ligands. These proteins bind to specific DNA sequences, termed hor- mone response elements (HREs), that reside close to hormone-responsive genes (reviewed In Ref. 1). This signal transduction process involves three discernible domains in the nuclear receptor proteins (reviewed in Refs. 2, 3)--the N-terminus, the central DNA-binding domain comprised of two zinc finger motifs, and the C- terminal ligand-binding domain; only the N-terminus is not well conserved. In general, virtually nothing is known about the origin of transcriptional regulatory factors. With respect to the nuclear receptor protefns in particular, hvo evolutionary histories have been proposed. The first assumes inde- pendent origins for the different domains. By this view, ligand-binding segments with functions in bioenergetics and intermediary metabolism bec.~e fused to a DNA- binding mot;f to produce transcription factors, and the ce~l acquired the ability to respond at the transcriptional revel to fluctuations in its physiological state (1). The second model implicates a s~gla, mult/-doma~n precur- sor that initia!Iy msd.~ated a ~s~rnp!e ~igna~ transduction mechanism {perh-~ps s:m;'.ar to that emp!oyed by t~e modem thyroid receptors) and subsequ~nt~j acqu::red increasingly complex functions (4, 5). Based on protein sequence comparison and evolutionary analysis, we believe the second model is correct. Our results sug- gest that all known nuclear receptors diverged from a single common ancestor. While this ancestor may have been formed by a domain-joining event, we find no evidence for subsequent exon-shuffllng or for homology between the nuclear receptors and any other transcrip- tion factors. We have investigated the evolution of the nuclear receptor gene family using established computer algo- rithms" to detect sequence similarity indicative of ho- moiogy (common evolutionary ancestry}. These ~tudies differ from searches for analogous sequences (6), which may share a common property (e.g. periodic cysteines and histidines) without sh~ng common ancestry. We used the amino acid sequences of the DNA-b=,n~ng domains (77 ~T~nO acid residues) from several nuclear receptors to search the PIR protein sequence database~ by Smith and Waterman (7) and FASTA analyses (8}=. In every case, the highest s~itarity scores betong~d to th-= nuclear receptor sequences in the database. For e×amp!e, the DNAobinding domain of th~ t FIR re'.e=..se 26, a~notat_KI a~d r~w s_~qu_=nces, 5.6 m:~.on re~:~ues in 19,451 entr;es. zp~j s~:'~uence comp~scns us.~d the PAM250 ~c~nng tSx (9) v~th a ~ena~ty ~f -12 for th~ fkst residue --4 for e.~~. a~nz~ re~u.= [n a 40O0OO14

A unique zinc finger protein is associated with active preferenti.ally . ecdysone responsive loci in Drosophila Sally A. Amero,t Sarah C.R. Elgin,2 and Ann L. Beyer1 IDepartment of Microbiology, University of ~rxrginia School of Medicine, Charlottesville, Virginia 22908 USA~ 2Departmen˘ of Biology, Washington Universi~/, St. Louis, Missouri 63130 USA Using an immunochemical approach, we have identified a unique antigen, PEP (protein on ecdysone puffs}, which is associated in third-instar larvae with the active ecdysone-regulated loci on polytene chromosomes; PEP is not associated with most intermolt puffs and is found on some, but not all, heat shock-induced puffs. The distribution pattern changes with changing puffing patterns in the developmental program. We have screened an expression library and recovered a cDNA clone encoding PEP..~EP possesses multiple potential nucleic acid- and protein- binding regions: a glycine- and asparagine-rlch amino terminus, four zinc finger motifs, two very acidic segments, two sho~t basic stretches, and an alanine- and proline-rich carboxyl terminus. The Pep gone maps by in situ hybridization to the cytological locus 74F, adjacent to the early ecdysone-responsive region; however, the gone is not regulated by ecdysone at the level of transcription. The pattern of Pep expression through development suggests that maternal Pep gone transcripts are supplied to the embryo, and that the abundance of Pep gone transcripts decreases to a lower, fairly constant level thereafter. This unusual protein may play a role in the process of gone activation, or possibly in RNA processing, for a defined set of developmentally regulated loci. [Key Words: Drosophfla~ ecdysone puffs~ locus 74F~ polytene chxomcsomes~ zinc fingers] Received October 3, 1990~ revised version accepted November 19, 1990. The giant polytene chromosomes of Drosophila melano- gaster provide a unique opportunity to assay the protein interactions at defined genetic loci, because specific pro- reins can be localized on the chromosomes through in- direct immunoehemical assays [e.g., Elgin et al. 1988). Analyses of this type have provided a general picture of transcriptional activity in puffs and interbands from the distribution patterns of KNA polymerase lI ISass 198Z~ Weeks etal. 1982) and topoisomerase I [Fleisehrnarm et al. 1984). The presence of heterogenous nuclear ribonu- eleoproteins, [hnRNPs} {S.A. Amero and A.L. Beyer, un- publ.] and small nuclear ribonucleoproteins [snRNPs) {Sass and Pederson 1984r S.A. Amero and A.L. Beyer, unpubl.), which are involved in packaging and processing of nascent KNA molecules {Dreytuss 1986~ Zieve and Sauterer 1990}, is also obsen, ed at these sites. A coordinated program of developmental gone expres- sion is visible as a reproducible, temporal pattern of puf/- ing activity on the polytene chromosomes [Ashbumer 1970). Three sets of chromosomal loci are included in the program, which is initiated in response to eedyster- oid hormones [Hodgetts etal. 1977]. The existing inter- molt puffs [including Io=i 25AC, 68C, and 90]~C1 regress~ several "early" puffs [including loci 2B, 22B, 23E, 63F, 74EF, and 75B) appear quie -l<ly and then regress. Subse- quentin5 >100 "late" pu~s appear in a teml~oral so- quence {Ashbumer and Berendes 1978~ Pongs 1988}. A number of early genes have been cloned [Burtis etal. 1990~ Segraves and Hogness 1990~ Thummel etal. 19901, and their products may psrtieipate in aetivatian of the late genes {Umess and Thummel 1990r for review, see Ashbumer 1990}~ the products of these late genes are thought to initiate metamorphosis. A different program of gone expression, and a different puffing pattern, ensues from heat shock or a number of other stresses {Ritossa 1962~ Ashbumer and Bonnet 1979}. Nine puffs, the sites of the genes encoding the heat shock proteins [Spradling etal. 1977), are rapidly induced at 37-380C~ eoncomitandy, the developmental puffs regress as transcription at these loci is repressed and splicing of pre-mRNAs is inhibited [Yost and Lindquist 1986]. At least one specific factor, the heat shock transcription factor, is required in addition to the basal t~anscriptional machinery for this induction pro- eess {Parker and Topoi 1984~ Wu 1984}. Using monoelonal antibodies to nuclear proteins from Drosopb21a embxvos, we have identified an unusual pro- rein, PEP [protein on ecdysone puff~], ~:-hich possesses a distinctive distribution pattern on polytene chromo- some.s: PEP is associated preferentially with activated, hormone-restmnsivc loci, including both early and late genes but is not Iound on most intermolt puffs, which 40000015