hnRNP-U is a specific DNA-dependent protein kinase substrate phosphorylated in response to DNA double-strand breaks

Cellular responses to DNA damage are orchestrated by the large phosphoinositol-3-kinase related kinases ATM, ATR and DNA-PK. We have developed a cell-free system to dissect the biochemical mechanisms of these kinases. Using this system, we identify heterogeneous nuclear ribonucleoprotein U (hnRNP-U), also termed scaffold attachment factor A (SAF-A), as a specific substrate for DNA-PK. We show that hnRNP-U is phosphorylated at Ser59 by DNA-PK in vitro and in cells in response to DNA double-strand breaks. Phosphorylation of hnRNP-U suggests novel functions for DNA-PK in the response to DNA damage.     

An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo

pEPI-1, a vector in which a chromosomal scaffold/matrix-attached region (S/MAR) is linked to the simian virus 40 origin of replication, is propagated episomally in CHO cells in the absence of the virally encoded large T-antigen and is stably maintained in the absence of selection pressure. It has been suggested that mitotic stability is provided by a specific interaction of this vector with components of the nuclear matrix. We studied the interactions of pEPI-1 by crosslinking with cis-diamminedichloroplatinum II, after which it is found to copurify with the nuclear matrix. In a south-western analysis, the vector shows exclusive binding to hnRNP-U/SAF-A, a multifunctional scaffold/matrix specific factor. Immunoprecipitation of the crosslinked DNA-protein complex demonstrates that pEPI-1 is bound to this protein in vivo. These data provide the first experimental evidence for the binding of an artificial episome to a nuclear matrix protein in vivo and the basis for understanding the mitotic stability of this novel vector class.     

Synergistic induction of the senescence-associated genes by 5-bromodeoxyuridine and AT-binding ligands in HeLa cells

5-Bromodeoxyuridine induces a senescence-like phenomenon in mammalian cells. This effect was dramatically potentiated by AT-binding ligands such as distamycin A, netropsin, and Hoechst 33258. The genes most remarkably affected by these ligands include the widely used senescence-associated genes and were located on or nearby Giemsa-dark bands of human chromosomes. We hypothesize that AT-rich scaffold/nuclear matrix attachment region sequences are involved in this phenomenon. In fact, upon substitution of thymine with 5-bromouracil, a rat S/MAR sequence reduced its degree of bending and became insensitive to cancellation of the bending by distamycin A. The S/MAR sequence containing 5-bromouracil also bound more tightly to nuclear scaffold proteins in vitro and this binding was not inhibited by distamycin A. Under the same conditions, the S/MAR sequence containing thymine easily dissociated from the nuclear scaffold proteins. Taken together, the synergistic induction of the genes may be explained not only by opening of condensed chromatin by distamycin A but also by increase in the binding of 5-bromouracil-containing S/MAR sequences to the nuclear scaffolds.     

The polyribosomal protein bound to the 3' end of histone mRNA can function in histone pre-mRNA processing.

Cell cycle-regulated histone mRNAs end in a conserved 26-nt sequence that can form a stem-loop with a six-base stem and a four-base loop. The 3' end of histone mRNA has distinct functions in the nucleus and in the cytoplasm. In the nucleus it functions in pre-mRNA processing and transport, whereas in the cytoplasm it functions in translation and regulation of histone mRNA stability. The stem-loop binding protein (SLBP), present in both nuclei and polyribosomes, is likely the trans-acting factor that binds to the 3' end of mature histone mRNA and mediates its function. A nuclear extract that efficiently processes histone pre-mRNA was prepared from mouse myeloma cells. The factor(s) that bind to the 3' end of histone mRNA can be depleted from this extract using a biotinylated oligonucleotide containing the conserved stem-loop sequence. Using this depleted extract which is deficient in histone pre-mRNA processing, we show that SLBP found in polyribosomes can restore processing, suggesting that SLBP associates with histone pre-mRNA in the nucleus, participates in processing, and then accompanies the mature mRNA to the cytoplasm.     

A bipartite U1 site represses U1A expression by synergizing with PIE to inhibit nuclear polyadenylation

U1A protein negatively autoregulates itself by polyadenylation inhibition of its own pre-mRNA by binding as two molecules to a 3'UTR-located Polyadenylation Inhibitory Element (PIE). The (U1A)2-PIE complex specifically blocks U1A mRNA biosynthesis by inhibiting polyA tail addition, leading to lower mRNA levels. U1 snRNP bound to a 5'ss-like sequence, which we call a U1 site, in the 3'UTRs of certain papillomaviruses leads to inhibition of viral late gene expression via a similar mechanism. Although such U1 sites can also be artificially used to potently silence reporter and endogenous genes, no naturally occurring U1 sites have been found in eukaryotic genes. Here we identify a conserved U1 site in the human U1A gene that is, unexpectedly, within a bipartite element where the other part represses the U1 site via a base-pairing mechanism. The bipartite element inhibits U1A expression via a synergistic action with the nearby PIE. Unexpectedly, synergy is not based on stabilizing binding of the inhibitory factors to the 3'UTR, but rather is a property of the larger ternary complex. Inhibition targets the biosynthetic step of polyA tail addition rather than altering mRNA stability. This is the first example of a functional U1 site in a cellular gene and of a single gene containing two dissimilar elements that inhibit nuclear polyadenylation. Parallels with other examples where U1 snRNP inhibits expression are discussed. We expect that other cellular genes will harbor functional U1 sites.     

An artificial riboswitch for controlling pre-mRNA splicing

Riboswitches, as previously reported, are natural RNA aptamers that regulate the expression of numerous bacterial metabolic genes in response to small molecule ligands. It has recently been shown that these RNA genetic elements are also present near the splice site junctions of plant and fungal introns, thus raising the possibility of their involvement in regulating mRNA splicing. Here it is shown for the first time that a riboswitch can be engineered to regulate pre-mRNA splicing in vitro. We show that insertion of a high-affinity theophylline binding aptamer into the 3' splice site (3' ss) region of a model pre-mRNA (AdML-Theo29AG) enables its splicing to be repressed by the addition theophylline. Our results indicate that the location of 3' ss AG within the aptamer plays a crucial role in conferring theophylline-dependent control of pre-mRNA splicing. We also show that theophylline-mediated control of pre-mRNA splicing is highly specific by first demonstrating that a small molecule ligand similar in shape and size to theophylline had no effect on the splicing of AdML-Theo29AG pre-mRNA. Second, theophylline failed to exert any influence on the splicing of a pre-mRNA that does not contain its binding site. Third, theophylline specifically blocks the step II of the splicing reaction. Finally, we provide evidence that theophylline-dependent control of pre-mRNA splicing is functionally relevant.     

Entamoeba histolytica: cloning and expression of the poly(A) polymerase EhPAP

In eukaryotes, polyadenylation of pre-mRNA 3' end is essential for mRNA export, stability, and translation. Here we identified and cloned a gene codifying for a putative nuclear poly(A) polymerase (EhPAP) in Entamoeba histolytica. Protein sequence alignments with eukaryotic PAPs showed that EhPAP has the RNA-binding region and the PAP central domain with the catalytic nucleotidyl transferase domain described for other nuclear PAPs. Recombinant EhPAP expressed in bacteria was used to generate specific antibodies, which recognized two EhPAP isoforms of 60 and 63kDa in nuclear and cytoplasmic extracts by Western blot assays. RT-PCR assays showed that EhPap mRNA expression varies in multidrug-resistant trophozoites growing in different emetine concentrations. Moreover, EhPap mRNA expression is about 10- and 7-fold increased in G1 and S phase, respectively, through cell cycle progression. These results suggest the existence of a link between EhPAP expression and MDR and cell cycle regulation, respectively.     

Characterization of SAF-A, a novel nuclear DNA binding protein from HeLa cells with high affinity for nuclear matrix/scaffold attachment DNA elements.

We identified four proteins in nuclear extracts from HeLa cells which specifically bind to a scaffold attachment region (SAR) element from the human genome. Of these four proteins, SAF-A (scaffold attachment factor A), shows the highest affinity for several homologous and heterologous SAR elements from vertebrate cells. SAF-A is an abundant nuclear protein and a constituent of the nuclear matrix and scaffold. The homogeneously purified protein is a novel double stranded DNA binding protein with an apparent molecular weight of 120 kDa. SAF-A binds at multiple sites to the human SAR element; competition studies with synthetic polynucleotides indicate that these sites most probably reside in the multitude of A/T-stretches which are distributed throughout this element. In addition we show by electron microscopy that the protein forms large aggregates and mediates the formation of looped DNA structures.     

前体mRNA的选择性多聚腺苷酸化与人类疾病

真核细胞的前体mRNA必须经过复杂的加工过程才能成熟,包括5’端加帽、剪接和3’端加工,其中3’加工包括3’端的切割和多聚腺苷酸化.该过程由前体mRNA上的顺式作用元件以及多个蛋白质因子控制.组成哺乳动物前体mRNA3’端加工机器的核心蛋白质复合体有切割和多聚腺苷酸化特异性因子、切割刺激因子、切割因子Ⅰ和切割因子Ⅱ.其他因子包括poly(A)聚合酶、poly(A)结合蛋白、偶对蛋白(symplekin)等.哺乳动物基因通常含有多个ploy(A)位点,选择性多聚腺苷酸化不仅可产生具有不同长度3’UTR的mRNA异构体,还可能改变基因的CDS区.作为真核生物基因表达调控的关键机制,选择性多聚腺苷酸化在细胞生长、增殖和分化中起着重要作用.本文综述了哺乳动物前体mRNA的3’端加工过程,3’端加工机器的组成及功能,探讨了选择性多聚腺苷酸化在多种人类疾病中的作用机制,以期为读者带来一些新的见解.     

A nucleolin-binding 3' untranslated region element stabilizes beta-globin mRNA in vivo

The normal expression of human beta globin is critically dependent upon the constitutively high stability of its encoding mRNA. Unlike with alpha-globin mRNA, the specific cis-acting determinants and trans-acting factors that participate in stabilizing beta-globin mRNA are poorly described. The current work uses a linker-scanning strategy to identify a previously unknown determinant of mRNA stability within the beta-globin 3' untranslated region (3'UTR). The new determinant is positioned on an mRNA half-stem opposite a pyrimidine-rich sequence targeted by alphaCP/hnRNP-E, a factor that plays a critical role in stabilizing human alpha-globin mRNA. Mutations within the new determinant destabilize beta-globin mRNA in intact cells while also ablating its 3'UTR-specific interaction with the polyfunctional RNA-binding factor nucleolin. We speculate that 3'UTR-bound nucleolin enhances mRNA stability by optimizing alphaCP access to its functional binding site. This model is favored by in vitro evidence that alphaCP binding is enhanced both by cis-acting stem-destabilizing mutations and by the trans-acting effects of supplemental nucleolin. These studies suggest a mechanism for beta-globin mRNA stability that is related to, but distinct from, the mechanism that stabilizes human alpha-globin mRNA.