Tristetraprolin (TTP) is a tandem CCCH zinc finger protein that was identified through its rapid induction by mitogens in fibroblasts. Studies of TTP-deficient mice and cells derived from them showed that TTP could bind to certain AU-rich elements in mRNAs, leading to increases in the rates of mRNA deadenylation and destruction. Known physiological target mRNAs for TTP include tumor necrosis factor alpha, granulocytemacrophage colony-stimulating factor, and interleukin-2␤. Here we used microarray analysis of RNA from wild-type and TTP-deficient fibroblast cell lines to identify transcripts with different decay rates, after serum stimulation and actinomycin D treatment. Of 250 mRNAs apparently stabilized in the absence of TTP, 23 contained two or more conserved TTP binding sites; nine of these appeared to be stabilized on Northern blots. The most dramatically affected transcript encoded the protein Ier3, recently implicated in the physiological control of blood pressure. The Ier3 transcript contained several conserved TTP binding sites that could bind TTP directly and conferred TTP sensitivity to the mRNA in cell transfection studies. These studies have identified several new, physiologically relevant TTP target transcripts in fibroblasts; these target mRNAs encode proteins from a variety of functional classes.

Summary Cells control dynamic transitions in transcript levels by regulating transcription, processing and/or degradation through an integrated regulatory strategy. Here, we combine RNA metabolic labeling, rRNA-depleted RNA-seq, and DRiLL, a novel computational framework, to quantify the level, editing sites, and transcription, processing and degradation rates of each transcript at a splice junction resolution during the LPS response of mouse dendritic cells. Four key regulatory strategies, dominated by RNA transcription changes, generate most temporal gene expression patterns. Non-canonical strategies that also employ dynamic posttranscriptional regulation control only a minority of genes, but provide unique signal processing features. We validate Tristetraprolin (TTP) as a major regulator of RNA degradation in one non-canonical strategy. Applying DRiLL to the regulation of non-coding RNAs and to zebrafish embryogenesis demonstrates its broad utility. Our study provides a new quantitative approach to discover transcriptional and post-transcriptional events that control dynamic changes in transcript levels using RNA-Seq data.

The MARCKS protein is a widely distributed cellular substrate for protein kinase C. It is a myristoylprotein that binds calmodulin and actin in a manner reversible by protein kinase C-dependent phosphorylation. It is also highly expressed in nervous tissue, particularly during development.

We isolated and sequenced a cDNA clone encoding the bovine "80-to 87-kDa" protein, a major cellular substrate for protein kinase C. An open reading frame of 1005 base pairs predicted a protein of 335 amino acids (Mr,31,949). Despite this predicted size, the protein migrated on SDS/ polyacrylamide gels with an apparent molecular weight of 80-87,000 after expression of the cDNA in cells lacking the protein.It was highly enriched in alanine (28.4 mol %), contained an amino-terminal myristoylation consensus sequence, and included a 25-residue basic domain containing the known protein kinase C phosphorylation sites. Two mRNA species (2.6 and 4.4 kilobases) were most highly expressed in brain, spinal cord, spleen, and lung, in parallel with the distribution of immunoreactive protein. Genomic blot analysis indicated the likelihood of a single gene coding for this mRNA. We propose the name myristoylated alanine-rich C kinase substrate (MARCKS) for this protein.Protein kinase C, the Ca2+/diacylglycerol-dependent protein kinase, is a ubiquitous enzyme known to consist of a family of related gene products (1, 2). Although protein kinase C itself is becoming better understood, little is known about its cellular substrates and how they are involved in the physiological processes affected by protein kinase C activation.One of the most prominent cellular substrates for the kinase, the "80-to 87-kDa protein", has received considerable attention in the past several years ( We undertook the present study to elucidate the structure of this protein and to characterize the sites phosphorylated by protein kinase C. In this paper, we describe the molecular cloning of a cDNA § encoding the bovine protein, and the use of this cDNA in studies of its mRNA expression. Since the predicted size of the protein bears little relationship to the size (80-87 kDa) observed on SDS/polyacrylamide gels and since amino acid analysis of the protein revealed nearly 30 mol % of alanine (7), we propose the name myristoylated alanine-rich C kinase substrate (MARCKS) for this protein. EXPERIMENTAL PROCEDURESProtein Purification, Tryptic Digestion, Purification of Peptides, Amino Acid Sequencing, and Synthesis and Labeling of Oligonucleotides and of cDNAs. The "80-to 87-kDa" protein was purified from bovine brain by the method of Albert et al. (7). Approximately 100 ug of protein was digested with trypsin and the tryptic peptides were separated by reversephase HPLC as described (8). Peaks of absorbance were monitored at 219 nm, and samples from several of the peaks were subjected to gas-phase sequencing on an Applied Biosystems model 470A sequencer in combination with a model 120A phenylthiohydantoin analyzer. Three peaks yielded useful peptide sequences (see Results). Six synthetic oligonucleotides were synthesized based on these amino acid sequences and were purified using C18 SEP-PAK columns (Millipore, Waters) as described in the Applied Biosystems DNA synthesizer user manual; these were labeled at the 5' end with [_y-32P]ATP (DuPont/NEN) using T...

Interruption of a single allele prevented formation of the subcommissural organ, a structure important for cerebrospinal fluid flow through the aqueduct of Sylvius, and resulted in congenital hydrocephalus. These data implicate the RFX4_v3 variant transcript as being crucial for early brain development, as well as for the genesis of the subcommissural organ. These findings may be relevant to human congenital hydrocephalus, a birth defect that affects ~0.6 per 1000 newborns.

The mouse gene Recql is a member of the RecQ subfamily of DEx-H-containing DNA helicases. Five members of this family have been identified in both humans and mice, and mutations in three of these, BLM, WRN, and RECQL4, are associated with human diseases and a cellular phenotype that includes genomic instability. To date, no human disease has been associated with mutations in RECQL and no cellular phenotype has been associated with its deficiency. To gain insight into the physiological function of RECQL, we disrupted Recql in mice. RECQL-deficient mice did not exhibit any apparent phenotypic differences compared to wild-type mice. Cytogenetic analyses of embryonic fibroblasts from the RECQL-deficient mice revealed aneuploidy, spontaneous chromosomal breakage, and frequent translocation events. In addition, the RECQL-deficient cells were hypersensitive to ionizing radiation, exhibited an increased load of DNA damage, and displayed elevated spontaneous sister chromatid exchanges. These results provide evidence that RECQL has a unique cellular role in the DNA repair processes required for genomic integrity. Genetic background, functional redundancy, and perhaps other factors may protect the unstressed mouse from the types of abnormalities that might be expected from the severe chromosomal aberrations detected at the cellular level.DNA helicases are ubiquitous enzymes that unwind DNA in an ATP-dependent and directionally specific manner. The unwinding of double-stranded DNA is essential for the processes of DNA repair, recombination, transcription, and DNA replication (1,19,30,35,45). One family of helicases is the RecQ subfamily of DEx-H-containing DNA helicases, of which Escherichia coli RecQ is the prototype member. All members of this family contain an approximately 450-amino-acid domain that contains seven helicase motifs of the DExH box superfamily (see Fig. S1 in the supplemental material). E. coli RecQ is a 3Ј35Ј helicase involved in the homologous recombination (HR) and double-strand break (DSB) repair mediated by the RecF pathway and suppression of illegitimate recombination (51). Other RecQ-related proteins include SGS1 in Saccharomyces cerevisiae, which plays a role in recombination, chromosome partitioning, and genome stability (32,53), and RQH1 in Schizosaccharomyces pombe, which is required to prevent recombination and suppression of inappropriate recombination (47).Unlike E. coli, S. cerevisiae, and S. pombe, which only have one RecQ helicase, certain bacteria (e.g., Bacillus subtilis [12] and Porphyromonas gingivalis [34]) and higher eukaryotes express multiple RecQ-related helicases (45). Five such helicases have been described in humans. The first described was RECQL or RECQL1 (39,40,43), followed by BLM (11, 44), WRN (59), RECQL4 (20,21), and RECQL5 (21

The mouse gene Zfp36L1 encodes zinc finger protein 36-like 1 (Zfp36L1), a member of the tristetraprolin (TTP) family of tandem CCCH finger proteins. TTP can bind to AU-rich elements within the 3-untranslated regions of the mRNAs encoding tumor necrosis factor (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), leading to accelerated mRNA degradation. TTP knockout mice exhibit an inflammatory phenotype that is largely due to increased TNF secretion. Zfp36L1 has activities similar to those of TTP in cellular RNA destabilization assays and in cell-free RNA binding and deadenylation assays, suggesting that it may play roles similar to those of TTP in mammalian physiology. To address this question we disrupted Zfp36L1 in mice. All knockout embryos died in utero, most by approximately embryonic day 11 (E11). Failure of chorioallantoic fusion occurred in about two-thirds of cases. Even when fusion occurred, by E10.5 the affected placentas exhibited decreased cell division and relative atrophy of the trophoblast layers. Although knockout embryos exhibited neural tube abnormalities and increased apoptosis within the neural tube and also generalized runting, these and other findings may have been due to deficient placental function. Embryonic expression of Zfp36L1 at E8.0 was greatest in the allantois, consistent with a potential role in chorioallantoic fusion. Fibroblasts derived from knockout embryos had apparently normal levels of fully polyadenylated compared to deadenylated GM-CSF mRNA and normal rates of turnover of this mRNA species, both sensitive markers of TTP deficiency in cells. We postulate that lack of Zfp36L1 expression during mid-gestation results in the abnormal stabilization of one or more mRNAs whose encoded proteins lead directly or indirectly to abnormal placentation and fetal death.The tristetraprolin (TTP) family of CCCH tandem zinc finger proteins consists of three known members in mammals and a fourth more distantly related protein in frogs and fish (reviewed in reference 2). The best-studied family member is TTP, which is the product of the immediate-early response gene Zfp36 in the mouse (ZFP36 in humans). TTP appears to act in normal physiology to bind to AU-rich elements (ARE) in certain mRNAs and to destabilize those transcripts. Specifically, the mRNAs encoding tumor necrosis factor (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are stabilized in TTP knockout (KO) mice and in cells derived from them. This results in excessive secretion of these cytokines from the appropriate cell types, resulting in a severe systemic inflammatory phenotype with myeloid hyperplasia in the TTP KO mice (37).The two other known mammalian TTP family members contain CCCH tandem zinc finger (TZF) domains that are very similar to that of TTP; these TZF domains represent the ARE-binding domains of the proteins. One of these proteins (Zfp36L1, for Zfp36-like 1) was actually the first member of this family in which the TZF domain was recognized (15). Its cDNA was originally cloned ...

The CCCH tandem zinc finger protein, Zfp36l2, like its better-known relative tristetraprolin (TTP), can decrease the stability of AU-rich element-containing transcripts in cell transfection studies; however, its physiological importance is unknown. We disrupted Zfp36l2 in mice, resulting in decreased expression of a truncated protein in which the N-terminal 29 amino acids had been deleted (DeltaN-Zfp36l2). Mice derived from different clones of ES cells exhibited complete female infertility, despite evidence from embryo and ovary transplantation experiments that they could gestate and rear wild-type young. DeltaN-Zfp36l2 females apparently cycled and ovulated normally, and their ova could be fertilized; however, the embryos did not progress beyond the two-cell stage of development. These mice represent a specific model of disruption of the earliest stages of embryogenesis, implicating Zfp36l2, a probable mRNA-binding and destabilizing protein, in the physiological control of female fertility at the level of early embryonic development. This newly identified biological role for Zfp36l2 may have implications for maternal mRNA turnover in normal embryogenesis, and conceivably could be involved in some cases of unexplained human female infertility.