The nuclear poly(A) binding protein of mammals, but not of fission yeast, participates in mRNA polyadenylation.

The nuclear poly(A) binding protein (PABPN1) has been suggested, on the basis of biochemical evidence, to play a role in mRNA polyadenylation by strongly increasing the processivity of poly(A) polymerase. While experiments in metazoans have tended to support such a role, the results were not unequivocal, and genetic data show that the S. pombe ortholog of PABPN1, Pab2, is not involved in mRNA polyadenylation. The specific model in which PABPN1 increases the rate of poly(A) tail elongation has never been examined in vivo. Here, we have used 4-thiouridine pulse-labeling to examine the lengths of newly synthesized poly(A) tails in human cells. Knockdown of PABPN1 strongly reduced the synthesis of full-length tails of ∼250 nucleotides, as predicted from biochemical data. We have also purified S. pombe Pab2 and the S. pombe poly(A) polymerase, Pla1, and examined their in vitro activities. Whereas PABPN1 strongly increases the activity of its cognate poly(A) polymerase in vitro, Pab2 was unable to stimulate Pla1 to any significant extent. Thus, in vitro and in vivo data are consistent in supporting a role of PABPN1 but not S. pombe Pab2 in the polyadenylation of mRNA precursors.

[Canonical and noncanonical RNA polyadenylation].

Polyadenylation is the non-template addition of adenosine nucleotides at the 3'-end of RNA, which occurs after transcription and generates a poly(A) tail up to 250-300 nucleotides long. In the first section of our review, we consider the classical process of mRNA 3'-terminus formation, which involves the cleavage of the transcript synthesized by RNA polymerase II and the associated poly(A) tail synthesis by canonical polyadenylate polymerase. Nucleotide sequences needed for mRNA cleavage and poly(A) tail synthesis, in particular the AAUAAA polyadenylation signal, as well as numerous proteins and their complexes involved in mRNA cleavage and polyadenylation, is described in detail. The significance of the poly(A) tail for prolonging mRNA lifetime and stimulating their translation is discussed. Data presented in the second section demonstrate that RNA transcribed by RNA polymerase III from certain SINEs (Short Interspersed Elements) can undergo AAUAAA-dependent polyadenylation. The structural and functional features of RNA polymerase III determine the unusual character of polyadenylation of RNAs synthesized by this enzyme. The history of recent developments in this area of study have been described in greater detail, in particular the discovery of AAUAAA-dependent polyadenylation of RNA synthesized by RNA polymerase III, which has not been discussed previously. Data on AAUAAA-independent polyadenylation catalyzed by noncanonical TRAMP poly(A)-polymerases (Trf4 and Trf5) have been presented in the third section. These enzymes promote rapid degradation of RNAs by adding a short poly(A) tail to them. This mechanism enables the recognition, poly(A)-marking, and elimination of incorrectly folded noncoding transcripts (e.g. ribosomal and transfer RNAs).

Multiple forms of chicken alpha 3(VI) collagen chain generated by alternative splicing in type A repeated domains.

Type VI collagen is a structurally unique component widely distributed in connective tissues. Its molecular structure consists of monomers that have the potential to assemble intracellularly into dimers and tetramers which, once secreted, can form microfilaments by end-to-end association. Individual monomers are composed of chains of Mr = approximately 140,000 (alpha 1 and alpha 2) and greater than 300,000 (alpha 3). Type VI collagen molecules contain a short triple helix with large globular domains at both ends. These domains are made for their greatest part of repetitive units similar to type A repeats of von Willebrand Factor. The alpha 3(VI) chain, contributing most of the mass of the NH2-terminal globule, appeared heterogenous both at the mRNA and protein level. Several alpha 3(VI)-specific clones that lack the sequences corresponding to repeats A8 and A6 were isolated from a chicken aorta cDNA library. Northern blot hybridization of poly (A+)-enriched RNA from chicken gizzard with cDNA fragments corresponding to several individual type A repeats showed that A8- and A6-specific probes did not hybridize to the lower Mr transcripts. Clones spanning approximately 20 kb of the 5'-end of the alpha 3(VI) gene were isolated from a chicken genomic library and subjected to analysis by restriction mapping, Southern blotting, and selective sequencing of the intron-exon boundaries. At the most 5'-end of the gene an additional type A repeat (A9), previously undetected in cDNA clones, was identified. Furthermore, it was determined that the presumed signal peptide and repeats A9 through A6 are encoded within individual exons. Reverse transcription and polymerase chain reaction of aorta RNA suggested that a mechanism of alternative mRNA splicing by a phenomenon of exon skipping generates alpha 3(VI) isoform variants that contain different numbers of type A repeats. Immunohistochemistry of frozen sections of chicken embryo tissues with repeat-specific mAbs showed that an antibody directed against a conditional exon has a more restricted tissue distribution compared to an antibody against a constitutive exon.

Single mRNAs visualized by ultrastructural in situ hybridization are principally localized at actin filament intersections in fibroblasts.

Considerable evidence indicates that mRNA associates with structural filaments in the cell (cytoskeleton). This relationship would be an important mechanism to effect mRNA sorting since specific mRNAs could be sequestered at sites within the cell. In addition, it can provide a mechanism for spatial regulation of mRNA expression. However, the precise structural interactions between mRNA and the cytoskeleton have yet to be defined. An objective of this work was to visualize "individual" poly(A) mRNA molecules in situ by electron microscopy to identify their relationship to individual filaments. Poly(A) RNA and filaments were identified simultaneously using antibodies to detect hybridized probe and filaments or actin-binding proteins. In human fibroblasts, most of the poly(A) mRNA (72%) was localized within 5 nm of orthogonal networks of F-actin filaments. Poly(A) mRNA also colocalized with vimentin filaments (29%) and microtubules (< 10%). The sites of mRNA localization were predominantly at filament intersections. The majority of poly(A) mRNA and polysomes colocalized with the actin crosslinking proteins, filamin, and alpha-actinin, and the elongation factor, EF-1 alpha (actin-binding protein; ABP-50). Evidence that intersections contained single mRNA molecules was provided by using a labeled oligo dT probe to prime the synthesis of cDNA in situ using reverse transcriptase. Both the poly(A) and cis sequences of the same mRNA molecule could then be visualized independently. We propose that the cytoskeletal intersection is a mRNA receptor and serves as a "microdomain" where mRNA is attached and functionally expressed.

Functional reconstitutional of the human epidermal growth factor receptor system in Xenopus oocytes.

We have expressed the human EGF receptor (hEGF-R) in Xenopus oocytes by injecting mRNA synthesized in vitro using SP6 vectors containing receptor cDNAs. Each oocyte could express over 1 x 10(10) receptors of a single affinity class and these were able to bind and rapidly internalize EGF. Occupancy resulted in receptor tyrosine autophosphorylation, downregulation, and release of intracellular calcium. Occupied receptors also rapidly induced meiotic maturation in stage VI oocytes. Receptors lacking tyrosine kinase activity bound EGF normally, but did not downregulate or induce any biological responses. The rate of oocyte maturation was proportional to hEGF-R occupancy and was significantly faster than progesterone-induced maturation at nanomolar EGF concentrations. Mutant hEGF-R truncated at residue 973 displayed identical phenotypes in both mammalian cells and oocytes in that they were defective in their ability to release intracellular calcium, undergo ligand induced internalization and receptor downregulation. However, these receptors were fully capable of inducing oocyte maturation. The remarkable retention of specific biological activities of different hEGF-R in the context of oocytes suggests that this receptor system interacts with generally available cellular components that have been conserved during evolution. In addition, it suggests that cell surface tyrosine kinase activity may play an important role in regulating resumption of the cell cycle.

How the messenger got its tail: addition of poly(A) in the nucleus.

Most mRNAs end in a poly(A) tail, the addition of which is catalysed by a poly(A) polymerase in conjunction with a distinct factor that provides specificity for mRNAs. The reaction is dynamic, involving separable initiation, elongation and termination phases. A companion article in next month's TIBS will review the regulation of poly(A) addition and removal during early animal development.

Cloning and localization of a conventional kinesin motor expressed exclusively in neurons.

Kinesin is a microtubule-based motor protein involved in organelle transport in neuronal and nonneuronal cells. Although a single kinesin motor has been thought to serve all cell types, we document here that neurons express a second conventional kinesin heavy chain (nKHC) that is 65% identical in amino acid sequence to the ubiquitously expressed kinesin heavy chain (uKHC). By preparing antibodies which distinguish between the two KHCs, we demonstrate that nKHC is a nucleotide-dependent microtubule-binding protein which partially cofractionates with membrane organelles. Immunolocalization experiments show that nKHC is distributed throughout the CNS but is highly enriched in subsets of neurons. In hippocampal neurons in culture, uKHC is distributed uniformly throughout the neuron, whereas nKHC is selectively concentrated in the cell body. These results demonstrate that mammalian neuronal tissue contains two conventional kinesin motors which may serve distinct functions in microtubule-based transport.

Blood-brain barrier genomics and cloning of a novel organic anion transporter.

A novel organic anion transporter selectively expressed at the blood-brain barrier (BBB), originally designated BBB-specific anion transporter type 1 (BSAT1), and now classified as Slco1c1, has been cloned from a BBB genomics program as a partial cDNA; this study describes the cloning and expression of the full-length cDNA from a rat brain capillary cDNA library. Northern analysis revealed the selective expression of the transporter at the BBB, and the transporter was expressed after permanent transfection of human 293 cells with cDNA encoding either the full length or open reading frame mRNA. The full-length transporter cDNA was 2.6 kb, and the mRNA was highly expressed at the rat brain microvasculature, but not in kidney, liver, heart, or lung, or in glial cells or brain glial tumors. Blood-brain barrier-specific anion transporter type 1 expression in 293 cells was poor after the transfection of the full-length cDNA, whereas transporter expression in 293 cells was high after transfection of the open reading frame. The transporter showed asymmetric kinetic properties in comparison of the influx and efflux of model substrates, thyroxine (T4), triiodothyronine (T3), and estradiol-glucuronide (E2G). Thyroxine and T3 inhibited the influx of E2G, but E2G did not inhibit thyroxine influx, and T3 only weakly inhibited the influx of T4. Extracellular E2G stimulated the transefflux of intracellular T4. Blood-brain barrier-specific anion transporter type 1 is a novel organic anion transporter that is a sodium-independent exchanger that may participate in the active efflux of iodothyronines and steroid conjugates at the BBB.

Polyadenylation of genomic RNA and initiation of antigenomic RNA in a positive-strand RNA virus are controlled by the same cis-element.

Genomes and antigenomes of many positive-strand RNA viruses contain 3'-poly(A) and 5'-poly(U) tracts, respectively, serving as mutual templates. Mechanism(s) controlling the length of these homopolymeric stretches are not well understood. Here, we show that in coxsackievirus B3 (CVB3) and three other enteroviruses the poly(A) tract is approximately 80-90 and the poly(U) tract is approximately 20 nt-long. Mutagenesis analysis indicate that the length of the CVB3 3'-poly(A) is determined by the oriR, a cis-element in the 3'-noncoding region of viral RNA. In contrast, while mutations of the oriR inhibit initiation of (-) RNA synthesis, they do not affect the 5'-poly(U) length. Poly(A)-lacking genomes are able to acquire genetically unstable AU-rich poly(A)-terminated 3'-tails, which may be generated by a mechanism distinct from the cognate viral RNA polyadenylation. The aberrant tails ensure only inefficient replication. The possibility of RNA replication independent of oriR and poly(A) demonstrate that highly debilitated viruses are able to survive by utilizing 'emergence', perhaps atavistic, mechanisms.

Regulation of lymphokine production and human T lymphocyte activation by 1,25-dihydroxyvitamin D3. Specific inhibition at the level of messenger RNA.

The steroid hormone, 1 alpha,25-dihydroxyvitamin D3 (calcitriol), has been shown to inhibit T cell proliferation, primarily through inhibition of interleukin 2 (IL-2) production. In these experiments, we show that calcitriol also markedly inhibited production of the lymphokine, gamma interferon (IFN-gamma), by activated human T lymphocytes. Regulation of both IL-2 and IFN-gamma production as well as transferrin receptor (TfR) expression by calcitriol was apparent at the messenger RNA (mRNA) level as determined by Northern blotting. The decrease in IL-2 and IFN-gamma mRNA that occurred with calcitriol treatment was coordinate and not apparent up to 12 h after phytohemagglutinin stimulation, whereas decreased accumulation of TfR mRNA was not present before 24-36 h. Furthermore, the effects of calcitriol on IL-2, IFN-gamma, and TfR mRNA accumulation were specific; actin mRNA accumulation was comparable between control and treated cells. These data indicate that calcitriol regulated proteins associated with T cell activation at the transcriptional level and that these effects were mediated in a specific, coordinate fashion.

The yeast CBP1 gene produces two differentially regulated transcripts by alternative 3'-end formation.

CBP1 is a yeast nuclear gene encoding a mitochondrial protein that stabilizes the 5' end of cytochrome b (cob) pre-mRNA. Cytochrome b is the only mitochondrially synthesized component of the respiratory chain complex III. Since the nuclearly encoded subunits of this complex are regulated at the transcriptional level by catabolite repression, we hypothesized that CBP1 might be similarly regulated. To test the idea that transcriptional regulation of CBP1 could coordinate an increase in cytochrome b mRNA stability with an increase in nuclearly encoded complex III subunit production, we characterized the change in abundance of CBP1 mRNA during derepression on a nonfermentable carbon source. Poly(A)+ RNA from derepressed yeast cells was examined by Northern (RNA) analyses with cRNA probes from CBP1. Both 2.2- and 1.3-kilobase (kb) transcripts were detected. The 1.3-kb mRNA lacked approximately 900 nucleotides of the 3' end of the 2.2-kb mRNA, which encodes the carboxyl-terminal 250 amino acid residues of the CBP1 coding sequence. Northern analyses of RNA isolated from deletion-insertion mutants of CBP1 and from strains that overexpress CBP1 mRNA demonstrated that both mRNAs were transcribed from the CBP1 gene. Furthermore, we demonstrated that the levels of the two CBP1 mRNAs were reciprocally regulated by the carbon source in the growth medium. This is the first description of a yeast gene from which two transcripts that can encode proteins with distinctly different coding properties are generated by alternative 3'-end formation.

Sequence and spatial requirements for the tissue- and species-independent 3'-end processing mechanism of plant mRNA.

Two cis-regulatory regions are required for efficient mRNA 3'-end processing of the maize 27-kDa zein mRNA: a region containing a duplicated AAUGAA poly(A) signal and a region that is present upstream from it. Strict spatial positioning of these two regions is required for efficient mRNA 3'-end processing. Insertion of a stuffer sequence as short as 17 or 18 bp either between the upstream region and the two AAUGAA motifs or between the two AAUGAA motifs drastically reduced the efficiency of 3'-end processing. Mutational analyses of the nucleotide preference at the fourth position of the AAUGAA motif revealed the preference order G > A >> C or U, suggesting that AAUAAA is neither a defective nor an optimal poly(A) signal for the 27-kDa zein mRNA. As for the 3' control region of the cauliflower mosaic virus (CaMV) transcription unit, the mRNA 3'-end processing mechanism mediated by the 27-kDa zein 3' control sequence is neither tissue nor species specific. The 3' upstream sequence of the 27-kDa zein gene can functionally replace that of the CaMV transcription unit. Conversely, the CaMV upstream sequence can mediate mRNA polyadenylation in the presence of a duplicated 27-kDa zein poly(A) signal. However, instead of the proximal poly(A) signal normally used in the 27-kDa zein mRNA, the distal signal is utilized. These results suggest that a general mechanism controls the 3'-end processing of plant mRNAs and that the cis-regulatory functions mediated by their upstream regions are interchangeable.

Repression of beta interferon gene expression in virus-infected cells is correlated with a poly(A) tail elongation.

Expression of beta interferon (IFN-beta) is transiently induced when Namalwa B cells (Burkitt lymphoma cell line) are infected by Sendai virus. In this study, we found that an elongation of the IFN-beta mRNA could be detected in virus-infected cells and that such a modification was not observed when the IFN-beta transcript was induced by a nonviral agent, poly(I-C). Treatment of the cells with a transcriptional inhibitor (actinomycin D or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) resulted in further elongation of the transcript. Characterization of the elongated IFN-beta transcript by primer extension and RNase H treatment showed that the modification was a result of an elongated poly(A) tail of up to 400 nucleotides. We conclude that the poly(A) tail elongation of the IFN-beta transcript is associated with the viral infection. Furthermore, the presence of the elongated IFN-beta transcript correlated with a decrease of IFN-beta protein in the medium and in cell extracts. Sucrose gradient analysis of cytoplasmic extracts showed that IFN-beta transcripts with elongated poly(A) tails were found in the nonpolysomal fractions, whereas the shorter transcripts could be detected in both polysomal and nonpolysomal fractions. A longer form of the IFN-beta mRNA was also found in the nonpolysomal fractions of cells not treated with transcriptional inhibitors. Thus, the observed regulation of IFN-beta mRNA is not entirely dependent on the inhibition of transcription. To our knowledge, this study provides the first example of a poly(A) tail elongation in somatic cells that negatively influences gene expression in vivo.

Temporal order of gene replication in Chinese hamster ovary cells.

To investigate the molecular basis of the regulatory mechanisms responsible for the orderly replication of the mammalian genome, we have developed an experimental system by which the replication order of various genes can be defined with relative ease and precision. Exponentially growing CHO-K1 cells were separated into populations representing various stages of the cell cycle by centrifugal elutriation and analyzed for cell cycle status flow cytometry. The replication of specific genes in each elutriated fraction was measured by labeling with 5-mercuri-dCTP and [3H]dTPP under conditions of optimal DNA synthesis after cell permeabilization with lysolecithin. Newly synthesized mercurated DNA from each elutriated fraction was purified by affinity chromatography on thiol-agarose and replicated with the large fragment of Escherichia coli DNA polymerase I by using [alpha-32P]dATP and random primers. The 32P-labeled DNA representative of various stages of the cell cycle was then hybridized with dot blots of plasmid DNA containing specific cloned genes. From these results, it was possible to deduce the nuclear DNA content at the time each specific gene replicated during S phase (C value). The C values of 29 genes, which included single-copy genes, multifamily genes, oncogenes, and repetitive sequences, were determined and found to be distributed over the entire S phase. Of the 28 genes studied, 19 had been examined by others using in vivo labeling techniques, with results which agreed with the replication pattern observed in this study. The replication times of nine other genes are described here for the first time. Our method of analysis is sensitive enough to determine the replication time of single-copy genes. The replication times of various genes and their levels of expression in exponentially growing CHO cells were compared. Although there was a general correlation between transcriptional activity and replication in the first half of S phase, examination of specific genes revealed a number of exceptions. Approximately 25% of total poly(A) RNA was transcribed from the late-replicating DNA.

Translational control of germ cell-expressed mRNA imposed by alternative splicing: opioid peptide gene expression in rat testis.

The three genes encoding the opioid peptide precursors (prodynorphin, proenkephalin, and proopiomelanocortin) are expressed in the rat testis. The sizes of the three opioid mRNAs in the testis differ from the sizes of the corresponding mRNAs in other rat tissues in which these genes are expressed. The smaller testicular proopiomelanocortin mRNA has previously been demonstrated to arise from alternative transcriptional initiation. In the present study, we found that the smaller testicular prodynorphin mRNA, expressed in Sertoli cells, results from alternative mRNA processing. Exon 2, which makes up 5' untranslated sequence, is removed from the mature transcript. Polysome analysis of brain and testis RNA indicates that the alteration of the prodynorphin leader sequence in the testis-specific transcript does not affect the efficiency of translation of this mRNA. The larger testicular proenkephalin transcript, expressed in developing germ cells, also results from alternative mRNA processing. Alternative acceptor site usage in the splicing of intron A results in a germ cell-specific proenkephalin transcript with a 491-nucleotide 5' untranslated leader sequence preceding the preproenkephalin-coding sequence. Polysome analysis indicates that this germ cell-specific proenkephalin mRNA is not efficiently translated. Mechanisms by which alternative mRNA splicing may serve to confer translational regulation upon the testicular proenkephalin transcript are discussed.

Accurate polyadenylation of procyclin mRNAs in Trypanosoma brucei is determined by pyrimidine-rich elements in the intergenic regions.

Polycistronic precursor RNAs from trypanosomes are processed into monocistronic mRNAs by the excision of intergenic sequences and the addition of a 39-nucleotide spliced leader by trans splicing. These mRNAs are also polyadenylated, yet they do not contain the hexamer AAUAAA within their 3' untranslated regions (UTRs). To identify the signals required for the accurate polyadenylation of mRNAs, we tested the effects of deletions in either the procyclin 3' UTR or the downstream intergenic region on the polyadenylation of transcripts from a reporter gene. Deletion of the entire 3' UTR does not affect polyadenylation, but a crucial element is located in the intergenic region and includes a pyrimidine-rich sequence from positions 79 to 112 followed by an AG dinucleotide. Related motifs are also found a similar distance downstream of other genes in both the procyclin and the variant surface glycoprotein expression sites. These sequences bear a strong resemblance to splice acceptor sites, but they are generally several hundred base pairs upstream of the major splice acceptor site of the next gene in the transcription unit. There is evidence, however, that some of them can give rise to alternatively spliced transcripts with unusually long 5' UTRs.

Multiple products from the shavenbaby-ovo gene region of Drosophila melanogaster: relationship to genetic complexity.

The Drosophila melanogaster shavenbaby (svb)-ovo gene region is a complex locus, containing two distinct but comutable genetic functions. ovo is required for survival and differentiation of female germ line cells and plays a role in germ line sex determination. In contrast, svb is required in both male and female embryos for the production of epidermal locomotor and sensory structures. Sequences required for the two genetic functions are partially overlapping. ovo corresponds to a previously described germ line-dependent 5.0-kb poly(A)+ mRNA that first appears in the germarium and accumulates in nurse cells during oogenesis. The 5.0-kb mRNA is stored in the egg, but it is rapidly lost in the embryos except for its continued presence in the germ line precursor pole cells. The ovo mRNA predicts a 1,028-amino-acid 110.6-kDa protein homologous with transcription factors. We have identified an embryonic mRNA, 7.1 kb in length, that contains exons partially overlapping those of the 5.0-kb poly(A)+ mRNA. The spatial distribution of this newly discovered transcript during midembryogenesis suggests that it corresponds to the svb function. The arrangement of exons common to the 5.0- and 7.1-kb mRNAs suggests that the Ovo and Svb proteins share DNA-binding specificity conferred by four Cys2-His2 zinc finger motifs but differ functionally in their capacity to interact with other components of the transcription machinery.

Amplification of the gene for histidyl-tRNA synthetase in histidinol-resistant Chinese hamster ovary cells.

Histidinol-resistant (HisOHR) mutants with up to a 30-fold increase in histidyl-tRNA synthetase activity have been isolated by stepwise adaptation of wild-type Chinese hamster ovary (CHO) cells to increasing amounts of histidinol in the medium. Immunoprecipitation of [35S]methionine-labeled cell lysates with antibodies to histidyl-tRNA synthetase showed increased synthesis of the enzyme in histidinol-resistant cells. The histidinol-resistant cell lines had an increase in translatable polyadenylated mRNA for histidyl-tRNA synthetase. A cDNA for CHO histidyl-tRNA synthetase has been cloned, using these histidyl-tRNA synthetase-overproducing mutants as the source of mRNA. Southern blot analysis of wild-type and histidinol-resistant cells with this cDNA showed that the histidyl-tRNA synthetase DNA bands were amplified in the resistant cells. These HisOHR cells owed their resistance to histidinol to amplification of the gene for histidyl-tRNA synthetase.

Polyadenylation of mRNA: minimal substrates and a requirement for the 2' hydroxyl of the U in AAUAAA.

mRNA-specific polyadenylation can be assayed in vitro by using synthetic RNAs that end at or near the natural cleavage site. This reaction requires the highly conserved sequence AAUAAA. At least two distinct nuclear components, an AAUAAA specificity factor and poly(A) polymerase, are required to catalyze the reaction. In this study, we identified structural features of the RNA substrate that are critical for mRNA-specific polyadenylation. We found that a substrate that contained only 11 nucleotides, of which the first six were AAUAAA, underwent AAUAAA-specific polyadenylation. This is the shortest substrate we have used that supports polyadenylation: removal of a single nucleotide from either end of this RNA abolished the reaction. Although AAUAAA appeared to be the only strict sequence requirement for polyadenylation, the number of nucleotides between AAUAAA and the 3' end was critical. Substrates with seven or fewer nucleotides beyond AAUAAA received poly(A) with decreased efficiency yet still bound efficiently to specificity factor. We infer that on these shortened substrates, poly(A) polymerase cannot simultaneously contact the specificity factor bound to AAUAAA and the 3' end of the RNA. By incorporating 2'-deoxyuridine into the U of AAUAAA, we demonstrated that the 2' hydroxyl of the U in AAUAAA was required for the binding of specificity factor to the substrate and hence for poly(A) addition. This finding may indicate that at least one of the factors involved in the interaction with AAUAAA is a protein.

Mammalian genes coordinately regulated by growth arrest signals and DNA-damaging agents.

More than 20 different cDNA clones encoding DNA-damage-inducible transcripts in rodent cells have recently been isolated by hybridization subtraction (A. J. Fornace, Jr., I. Alamo, Jr., and M. C. Hollander, Proc. Natl. Acad. Sci. USA 85:8800-8804, 1988). In most cells, one effect of DNA damage is the transient inhibition of DNA synthesis and cell growth. We now show that five of our clones encode transcripts that are increased by other growth cessation signals: growth arrest by serum reduction, medium depletion, contact inhibition, or a 24-h exposure to hydroxyurea. The genes coding for these transcripts have been designated gadd (growth arrest and DNA damage inducible). Two of the gadd cDNA clones were found to hybridize at high stringency to transcripts from human cells that were induced after growth cessation signals or treatment with DNA-damaging agents, which indicates that these responses have been conserved during mammalian evolution. In contrast to results with growth-arrested cells that still had the capacity to grow after removal of the growth arrest conditions, no induction occurred in HL60 cells when growth arrest was produced by terminal differentiation, indicating that only certain kinds of growth cessation signals induce these genes. All of our experiments suggest that the gadd genes are coordinately regulated: the kinetics of induction for all five transcripts were similar; in addition, overexpression of gadd genes was found in homozygous deletion c14CoS/c14CoS mice that are missing a small portion of chromosome 7, suggesting that a trans-acting factor encoded by a gene in this deleted portion is a negative effector of the gadd genes. The gadd genes may represent part of a novel regulatory pathway involved in the negative control of mammalian cell growth.