The La protein is an essential RNA-binding protein implicated in different aspects of RNA metabolism. Herein, we report that small interfering (siRNA)-mediated La depletion reduces cell proliferation of different cell lines concomitant with a reduction in cyclin D1 (CCND1) protein. To exclude off-target effects we demonstrate that exogenous La expression in La-depleted cells restores cell proliferation and CCND1 protein levels. In contrast, proliferation of immortalized CCND1 knockout cells is not affected by La depletion, supporting a functional coherence between La, CCND1 and proliferation. Furthermore, we document by reversible in vivo crosslinking and ribonucleoprotein (RNP) immunoprecipitation an association of the La protein with CCND1 messengerRNA and that CCND1 internal ribosome entry site (IRES)-dependent translation is modulated by La protein level within the cell. In addition, we show elevated La protein expression in cervical cancer tissue and its correlation with aberrant CCND1 protein levels in cervical tumor tissue lysates. In conclusion, this study establishes a role of La in cell proliferation and CCND1 expression and demonstrates for the first time an overexpression of the RNA-binding protein La in solid tumors.

A surprisingly large population of mRNAs has been shown to localize to sensory axons, but few RNA-binding proteins have been detected in these axons. These axonal mRNAs include several potential binding targets for the La RNA chaperone protein. La is transported into axonal processes in both culture and peripheral nerve. Interestingly, La is posttranslationally modified in sensory neurons by sumoylation. In axons, small ubiquitin-like modifying polypeptides (SUMO)-La interacts with dynein, whereas native La interacts with kinesin. Lysine 41 is required for sumoylation, and sumoylation-incompetent La K41R shows only anterograde transport, whereas WT La shows both anterograde and retrograde transport in axons. Thus, sumoylation of La determines the directionality of its transport within the axonal compartment, with SUMO-La likely recycling to the cell body.axonal transport ͉ La/SSB ͉ RNA localization ͉ small ubiquitin-like modifying polypeptide

Here we provide evidence for an interaction-dependent subnuclear trafficking of the human La (hLa) protein, known as transient interaction partner of a variety of RNAs. Among these, precursor transcripts of certain RNAs are located in the nucleoplasm or nucleolus. Here we examined which functional domains of hLa are involved in its nuclear trafficking. By using green fluorescent-hLa fusion proteins, we discovered a nucleolar localization signal and demonstrated its functionality in a heterologous context. In addition, we revealed that the RRM2 motif of hLa is essential both for its RNA binding competence in vitro and in vivo and its exit from the nucleolus. Our data imply that hLa traffics between different subnuclear compartments, which depend decisively on a functional nucleolar localization signal as well as on RNA binding. Directed trafficking of hLa is fully consistent with its function in the maturation of precursor RNAs located in different subnuclear compartments.The human La protein (hLa) 1 is a 47-kDa phosphoprotein predominantly localized in the nucleus. It was first discovered as an autoantigen recognized by antibodies present in the sera of patients suffering from systemic lupus erythematosus and Sjogren's syndrome (1, 2). The La protein is a member of a large group of RNA-binding proteins containing RNA recognition motifs (RRMs) (3-7) and interacts with a variety of small RNAs (for reviews see Refs. 8 -10). The La protein is implicated in several aspects of the RNA metabolism, including processing of precursors of tRNAs, U3 snoRNA, U1 RNA, U6 snRNA (11)(12)(13)(14)(15)(16)(17)(18)(19)(20), and stabilization of viral (21-23) as well as cellular RNAs (24). In the cytoplasm, La has been implicated in the translational control of viral (25-27) and cellular RNAs (28 -30).The human La protein contains a nuclear localization sequence at the very C-terminal end, and the staining for endogenous La as well as the distribution of GFP-tagged La reveal a predominantly diffuse nuclear staining, although this may appear different under certain conditions or treatments (31-33). Subcellular distribution of human and yeast La was assumed to be independent of phosphorylation (34, 35); however, recent studies (36, 37) revealed that phosphorylation at Ser-366 may well influence subcellular localization of La. A regulated, nonhomogeneous nuclear distribution of La may also be predicted from the distinct nuclear distribution of its various RNA interaction partners. For instance, pre-tRNAs are most likely synthesized in the nucleoplasm (38) but are processed, at least in part, in the nucleolus (39, 40), whereas in yeast the La-associated precursor of U3 snoRNA is probably processed in the nucleolar body and nucleolus (20). Nevertheless, interactions with precursor RNAs in different nuclear compartments implicate a highly dynamic and regulated nuclear distribution of La. Although a nucleolar localization of La has been reported (for review see Ref. 9), the hLa protein was not identified among the recently resolved protein ...

Survival time-associated plant homeodomain (PHD) finger protein in Ovarian Cancer 1 (SPOC1, also known as PHF13) is known to modulate chromatin structure and is essential for testicular stem-cell differentiation. Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after γ-irradiation (γ-IR), non-homologous end-joining (NHEJ) repair activity, and cellular radioresistance, but impairs homologous recombination (HR) repair. Conversely, SPOC1 overexpression delays IRIF formation and γH2AX expansion, reduces NHEJ repair activity and enhances cellular radiosensitivity. SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-α and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1. In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation. These findings provide the first evidence for a function of SPOC1 in DNA damage response (DDR) and repair. SPOC1 acts as a modulator of repair kinetics and choice of pathways. This involves its dose-dependent effects on DNA damage sensors, repair mediators and key regulators of chromatin structure.

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The La protein was recently identified as a host factor potentially involved in the cytokine-induced post-transcriptional down-regulation of hepatitis B virus (HBV) RNA. The La binding site was mapped to a predicted stem-loop structure within a region shared by all HBV RNAs, and it was concluded that the La protein might be an HBV RNA-stabilizing factor. To characterize the RNA binding mediated by the different RNA recognition motifs (RRMs) of the human La protein, several La deletion mutants were produced and analyzed for HBV RNA binding ability. The data demonstrate that the first RRM is not required for binding, whereas the RNP-1 and RNP-2 consensus sequences of the RRM-2 and RRM-3 are separately required for binding, indicating a cooperative function of these two RRMs. Furthermore, the results suggest that multimeric La disassembles into monomeric La upon binding of HBV RNA.B. By gel retardation assay the affinity of the wild type human La⅐HBV RNA.B interaction was determined in the nanomolar range, comparable to the affinity determined for the mouse La⅐HBV RNA.B interaction. This study identified small regions within the human La protein mediating the binding of HBV RNA. Hence, these binding sites might represent targets for novel antiviral strategies based on the disruption of the human La⅐HBV RNA interaction, thereby leading to HBV RNA degradation.The human La protein is a 47-kDa phosphoprotein predominantly localized in the nucleus. It was first discovered as an autoantigen recognized by antibodies present in sera of patients suffering from systemic lupus erythematosus and Sjögren's syndrome (1, 2). The La protein is a member of a large group of RNA-binding proteins containing RNA recognition motifs (RRM) 1 (3-8) and is implicated in several steps of RNA metabolism. Among the different La proteins identified in a variety of organisms, the N-terminal part is highly conserved (9). La was shown to co-immunoprecipitate with a number of small RNA molecules (10). A role for La in the termination of RNA polymerase III transcription has been described. It was shown that La interacts with RNA polymerase III transcripts such as pre-tRNA by binding to a small stretch of uridines at the 3Ј-end common to these transcripts and might be necessary for proper processing of these precursors (11-17). In addition, La is known to interact with a variety of viral and other cellular . La is also suggested to be involved in the capindependent translation initiation of several viruses, including polio virus and hepatitis C virus (19,(27)(28)(29), and more recently evidence is growing that La stabilizes various RNAs, such as histone and hepatitis C and B virus RNA (22,23,25,30,31). At this time point it is not clear yet how La fulfills all of these different functions, however, assuming that this protein acts as a RNA chaperone, thereby stabilizing RNA structures, a function in these varied processes might be envisaged.The human La protein contains three RNA recognition motifs (RRM) involved in the binding of RNAs (9), although t...

The La protein is a multifunctional RNA-binding protein and has also been suggested to be involved in the stabilization of hepatitis B virus (HBV) RNA. Here we demonstrate that antibodies against the human La protein specifically precipitate HBV RNA from HBV ribonucleoprotein-containing mammalian cell extracts, providing evidence for the association between human La and HBV RNA. Moreover, we report that the turnover of HBV RNA depends on structural features and less on the primary sequence of the La-binding site on the viral RNA. In addition we show that the interaction between human La and HBV RNA in vitro is modulated by accessory factor(s) in a phosphorylation-dependent manner. Taken together these data indicate that both structural features, the composition of La/HBV ribonucleoprotein particles as well as interacting cellular factors, are critical determinants in the regulation of the stability of the HBV RNA.RNA metabolism depends on the formation of ribonucleoprotein particles mediating diverse processes such as splicing, polyadenylation, nuclear export, and the regulation of mRNA stability (1, 2). The formation of RNPs 1 is a tightly controlled process, potentially regulated by several stimuli, including hormones and cytokines. Such stimuli can alter the RNA binding activity of proteins on the post-translational level by phosphorylation or dephosphorylation and thereby the processing and stability of RNAs (3-5). In addition, RNA processing depends on various cis-acting elements including splice sites, export elements, and endoribonucleolytic cleavage sites recognized by RNA-binding proteins. To understand fully the regulation of processing of a specific RNA, both trans-acting factors and cis-acting elements as well as their functions need to be known. The same applies for a detailed understanding of the metabolism of viral RNA. Such studies could lead to the identification of novel cellular targets valuable for the development of innovative antiviral strategies when focused on the post-transcriptional control of RNAs of viruses with global medical importance. This applies to hepatitis B virus (HBV) with more than 300 million chronically infected carriers worldwide who await more effective antiviral therapies.HBV is a noncytopathic, hepatotropic virus with a 3.2-kb circular DNA genome. After conversion into a covalently closed circular DNA, this genome serves in the nucleus as a template for transcription of all viral RNAs. Synthesis of these transcripts is driven by at least four promotors, leading to a large size heterogeneity with many different 5Ј-ends, whereas they all have very similar 3Ј-ends because of processing at the same polyadenylylation site (6). The so-called pregenomic RNA (slightly longer than genome length) is encapsidated into nucleocapsids where it is reverse-transcribed into viral DNA. This RNA serves also as messenger for synthesis of the viral P protein as well as for the core protein. The viral surface proteins as well as a regulatory protein with a role in hepatocarcinogenesis, des...

Hepatitis B virus (HBV) is hyperendemic to southern Africa, with genotype A of HBV being the predominant genotype, and subgenotype A1 prevailing. Infection with this subgenotype is associated with rapid disease progression, and high frequency of hepatocellular carcinoma development. The objectives of our study was to construct recombinant 1.28 mer replication competent HBV DNA plasmids of subgenotypes A1, A2 and D3 containing authentic endogenous HBV promoters and to follow their replication in vitro after transfection of Huh7 cells. We found that subgenotype D3 replicated at a lower level, as measured by HBsAg and HBV DNA levels, when compared to cells transfected with genotype A. There was no difference in the intracellular and extracellular HBsAg between cells transfected with subgenotypes A1 or A2. Cells transfected with subgenotype A1 had higher levels of intracellular replicative intermediates and HBcAg, and lower extracellular expression of HBeAg from days 1 to 3, when compared to cells transfected with subgenotype A2. In conclusion, the generation of these replication competent clones is an important step in the functional characterization of subgenotypes of HBV circulating in Africa and their comparison to strains circulating in other geographical regions of the world.