DDX3 modulates cell adhesion and motility and cancer cell metastasis via Rac1-mediated signaling pathway.

The DEAD-box RNA helicase DDX3 is a versatile protein involved in multiple steps of gene expression and various cellular signaling pathways. DDX3 mutations have been implicated in the wingless (Wnt) type of medulloblastoma. We show here that small interfering RNA-mediated DDX3 knockdown in various cell lines increased cell-cell adhesion but decreased cell-extracellular matrix adhesion. Moreover, DDX3 depletion suppressed cell motility and impaired directional migration in the wound-healing assay. Accordingly, DDX3-depleted cells exhibited reduced invasive capacities in vitro as well as reduced metastatic potential in mice. We also examined the mechanism underlying DDX3-regulated cell migration. DDX3 knockdown reduced the levels of both Rac1 and ß-catenin proteins, and consequentially downregulated the expression of several ß-catenin target genes. Moreover, we demonstrated that DDX3-regulated Rac1 mRNA translation, possibly through an interaction with its 5'-untranslated region, and affected ß-catenin protein stability in an Rac1-dependent manner. Taken together, our results indicate the DDX3-Rac1-ß-catenin regulatory axis in modulating the expression of Wnt/ß-catenin target genes. Therefore, this report provides a mechanistic context for the role of DDX3 in Wnt-type tumors.

Transportin-SR2 mediates nuclear import of phosphorylated SR proteins.

Serine/arginine-rich proteins (SR proteins) are a family of nuclear factors that play important roles in both constitutive and regulated precursor mRNA splicing. The domain rich in arginine/serine (RS) repeats (RS domain) serves as both a nuclear and subnuclear localization signal. We previously identified an importin beta family protein, transportin-SR2 (TRN-SR2), that specifically interacts with phosphorylated RS domains. A TRN-SR2 mutant deficient in Ran binding colocalizes with SR proteins in nuclear speckles, suggesting a role of TRN-SR2 in nuclear targeting of SR proteins. Using in vitro import assays, we here show that nuclear import of SR protein fusions requires cytosolic factors, and that the RS domain becomes phosphorylated in the import reaction. Reconstitution of SR protein import by using recombinant transport factors clearly demonstrates that TRN-SR2 is capable of targeting phosphorylated, but not unphosphorylated, SR proteins to the nucleus. Therefore, RS domain phosphorylation is critical for TRN-SR2-mediated nuclear import. Interestingly, we found that the RNA-binding activity of SR proteins confers temperature sensitivity to their nuclear import. Finally, we show that TRN-SR2 interacts with a nucleoporin and is targeted not only to the nuclear envelope but also to nuclear speckles in vitro. Thus, TRN-SR2 may perhaps escort SR protein cargoes to nuclear subdomains.

A human importin-beta family protein, transportin-SR2, interacts with the phosphorylated RS domain of SR proteins.

Serine/arginine-rich proteins (SR proteins) are mainly involved in the splicing of precursor mRNA. RS domains are also found in proteins that have influence on other aspects of gene expression. Proteins that contain an RS domain are often located in the speckled domains of the nucleus. Here we show that the RS domain derived from a human papillomavirus E2 transcriptional activator can target a heterologous protein to the nucleus, as it does in many other SR proteins, but insufficient for localization in speckles. By using E2 as a bait in a yeast two-hybrid screen, we identified a human importin-beta family protein that is homologous to yeast Mtr10p and almost identical to human transportin-SR. This transportin-SR2 (TRN-SR2) protein can interact with several cellular SR proteins. More importantly, we demonstrated that TRN-SR2 can directly interact with phosphorylated, but not unphosphorylated, RS domains. Finally, an indirect immunofluoresence study revealed that a transiently expressed TRN-SR2 mutant lacking the N-terminal region becomes localized to the nucleus in a speckled pattern that coincides with the distribution of the SR protein SC35. Thus, our results likely reflect a role of TRN-SR2 in the cellular trafficking of phosphorylated SR proteins.

A human papillomavirus E2 transcriptional activator. The interactions with cellular splicing factors and potential function in pre-mRNA processing.

The human papillomavirus (HPV) E2 protein plays an important role in transcriptional regulation of viral genes as well as in viral DNA replication. Unlike most types of HPV, the E2 protein of epidermodysplasia verruciformis (EV)-associated HPVs harbors a relatively long hinged region between the terminal, conserved transactivation and DNA binding/dimerization domains. The sequence of EV-HPV E2 hinge contains multiple arginine/serine (RS) dipeptide repeats which are characteristic of a family of pre-messenger RNA splicing factors, called SR proteins. Here we show that the HPV-5 (an EV-HPV) E2 protein can specifically interact with cellular splicing factors including a set of prototypical SR proteins and two snRNP-associated proteins. Transiently expressed HPV-5 E2 protein colocalizes with a nuclear matrix associated-splicing coactivator in nuclear speckled domains. The RS-rich hinge is essential for E2 transactivator interaction with splicing factors and for its subnuclear localization. Moreover, we present functional evidence for the HPV-5 E2 transactivator, which shows that the RS-rich hinge domain of the E2 protein can facilitate the splicing of precursor messenger RNA made via transactivation by E2 itself. Our results, therefore, suggest that a DNA binding transactivator containing an RS-rich sequence can play a dual role in gene expression.

Pre-mRNA splicing: the discovery of a new spliceosome doubles the challenge.

A rare class of pre-mRNA introns with non-canonical consensus sequences has been identified in metazoan genes. The novel, low-abundance spliceosome that excises these introns contains one small nuclear ribonucleoprotein (snRNP) in common with the major spliceosome (U5) and four snRNPs that are distinct from, but structurally and functionally analogous to U1, U2 and U4-U6. The architecture of RNA components at the presumptive core of the AT-AC splicesome supports current models of the spliceosomal active center and raises tantalizing questions about spliceosome evolution.

More Sm snRNAs from vertebrate cells.

There are a number of low-abundance small nuclear RNAs (snRNAs) in eukaryotic cells. Many of them have been assigned functions in the biogenesis of cellular RNAs, such as splicing and 3' end processing. Here, we present the sequence of Xenopus U12 snRNA and compare the secondary structures of the low-abundance U11 and U12 with those of the high-abundance U1 and U2, respectively. The data suggest functional parallels between these two pairs of snRNAs in pre-mRNA splicing. Using a highly sensitive method, we have identified several new low-abundance snRNAs from HeLa cells. These include five U7 snRNA variants and six novel snRNAs. One of the six novel RNAs is an Sm snRNA, whereas the rest are not immunoprecipitable by either anti-Sm antibodies or anti-trimethylguanosine antibodies. The discovery of these new RNAs suggests that there may be yet more low-abundance snRNAs in the nuclei of eukaryotic cells.

Highly diverged U4 and U6 small nuclear RNAs required for splicing rare AT-AC introns.

Removal of a rare class of metazoan precursor messenger RNA introns with AU-AC at their termini is catalyzed by a spliceosome that contains U11, U12, and U5 small nuclear ribonucleoproteins. Two previously unidentified, low-abundance human small nuclear RNAs (snRNAs), U4atac and U6atac, were characterized as associated with the AT-AC spliceosome and necessary for AT-AC intron splicing. The excision of AT-AC introns therefore requires four snRNAs not found in the major spliceosome. With the use of psoralen crosslinking, a U6atac interaction with U12 was identified that is similar to a U6-U2 helix believed to contribute to the spliceosomal active center. The conservation of only limited U6atac sequences in the neighborhood of this interaction and the potential of U6atac to base pair with the 5' splice site consensus for AT-AC introns provide support for current models of the core of the spliceosome.

A novel spliceosome containing U11, U12, and U5 snRNPs excises a minor class (AT-AC) intron in vitro.

A minor class of introns with noncanonical splice (AT-AC) and branch site sequences exists in metazoan protein coding genes. We have established a HeLa cell in vitro system that accurately splices a pre-mRNA substrate containing such an intron from the human P120 gene. Splicing occurs via a lariat intermediate whose branch site A residue is predicted to bulge from a duplex formed with the low abundance U12 small nuclear ribonucleoprotein (snRNP), which we confirm by psoralen cross-linking. Native gel electrophoresis reveals that U11, U12, and U5 snRNPs assemble onto the P120 pre-mRNA to form splicing complexes. Inhibition of P120 splicing by 2'-O-methyl oligonucleotides complementary to U12 or U5 demonstrates that U12 and U5 snRNPs perform essential roles in the AT-AC spliceosome.

Site-specific substitution of inosine at the terminal positions of a pre-mRNA intron: implications for the configuration of the terminal base interaction.

Genetic evidence in yeast has revealed that a non-Watson-Crick base-pairing interaction between terminal guanosine residues of the intron is required for the second step of pre-mRNA splicing. To explore the likely configuration of the interaction between the terminal guanosines of the intron, inosine was uniformly incorporated into an adenovirus pre-mRNA substrate (Ade) to replace guanosine residues. Splicing of the inosine-containing Ade pre-mRNA was completely inhibited. Psoralen cross-linking reveals that the association of U1 and U2 snRNPs with the intron was impaired. To eliminate the deleterious effects caused by complete inosine replacement, guanosine residues at the splice site(s) of the Ade pre-mRNA were substituted by inosine. Such pre-mRNA substrates were obtained by ligation of two or three RNA fragments; the 3' piece was primed with inosine or mono-phosphate inosine. Splicing of the Ade pre-mRNA containing an inosine residue at the 5' or the 3' splice site, or at both sites proceeds normally. Thus, the functions of the terminal guanosine residues of the intron in splicing can be replaced by inosine. This result supports the previous notion that an N1-carbonyl symmetric interaction likely occurs between the intron terminal residues during pre-mRNA splicing.

U12 snRNA in vertebrates: evolutionary conservation of 5' sequences implicated in splicing of pre-mRNAs containing a minor class of introns.

A minor class of introns with noncanonical splice sites has been identified in both vertebrate and invertebrate genomes. The divergent consensus sequences within these introns suggest that splicing might be via a mechanism distinct from that used by the major class of introns. The low abundance U12 snRNA has been proposed to base pair with the predicted branch site sequence of these minor class introns, probably bulging out an adenosine to act as the nucleophile in the first step of splicing. We have identified homologues of the previously characterized human U12 snRNA in both mouse and chicken, where the minor class of introns has also been found. The U12 sequences that potentially base pair with the putative branch site are invariant. Additional conserved sequences at the 5' end of U12 snRNA could dynamically base pair with U6 snRNA sequences flanking the hexanucleotide ACAGAG to form structures analogous to those of three U2-U6 interactions genetically defined as important in the major class of spliceosome. We have also isolated two human U12 snRNA genes. One gene is functional for transcription of U12 snRNA, whereas the other appears to be a pseudogene. Sequences of the 3' box in both U12 snRNA genes are strikingly similar and bear high resemblance to those of U1 and U2 genes. Upstream elements, including the PSE and the DSE, have been identified and characterized in the functional gene. These features indicate that transcription of U12 snRNA is driven by RNA polymerase II.

Modulation of 5' splice site choice in pre-messenger RNA by two distinct steps.

Ser/Arg-rich proteins (SR proteins) are essential splicing factors that commit pre-messenger RNAs to splicing and also modulate 5' splice site choice in the presence or absence of functional U1 small nuclear ribonucleoproteins (snRNPs). Here, we perturbed the U1 snRNP in HeLa cell nuclear extract by detaching the U1-specific A protein using a 2'-O-methyl oligonucleotide (L2) complementary to its binding site in U1 RNA. In this extract, the standard adenovirus substrate is spliced normally, but excess amounts of SR proteins do not exclusively switch splicing from the normal 5' splice site to a proximal site (site 125 within the adenovirus intron), suggesting that modulation of 5' splice site choice exerted by SR proteins requires integrity of the U1 snRNP. The observation that splicing does not necessarily follow U1 binding indicates that interactions between the U1 snRNP and components assembled on the 3' splice site via SR proteins may also be critical for 5' splice site selection. Accordingly, we found that SR proteins promote the binding of the U2 snRNP to the branch site and stabilize the complex formed on a 3'-half substrate in the presence or absence of functional U1 snRNPs. A novel U2/U6/3'-half substrate crosslink was also detected and promoted by SR proteins. Our results suggest that SR proteins in collaboration with the U1 snRNP function in two distinct steps to modulate 5' splice site selection.

SR proteins can compensate for the loss of U1 snRNP functions in vitro.

SR proteins are essential splicing factors that also influence 5' splice site choice. We show that addition of excess mixed SR proteins to a HeLa in vitro splicing system stimulates utilization of a novel 5' splice site (site 125) within the intron of the standard adenovirus pre-mRNA substrate. When U1 snRNPs are debilitated by sequestering the 5' end of U1 snRNA with a 2'-O-methyl oligoribonucleotide, excess SR proteins not only rescue splicing at the normal site and site 125 but also activate yet another 5' splice site (site 47) in the adenovirus intron. One SR protein, SC35, is sufficient to exhibit the above activities. The possibility that excess SR proteins recruit residual unblocked U1 snRNPs to participate in 5' splice site recognition has been ruled out by psoralen cross-linking studies, which demonstrate that the 2'-O-methyl oligoribonucleotide effectively blocks 5' splice site/U1 interaction. Native gel analysis reveals a nearly normal splicing complex profile in the 2'-O-methyl oligoribonucleotide pretreated, SR protein-supplemented extract. These results indicate that SR proteins can replace some functions of the U1 snRNP but underscore the contribution of U1 to the fidelity of 5' splice site selection.

Functional association of essential splicing factor(s) with PRP19 in a protein complex.

We have previously shown that the yeast PRP19 protein is a spliceosomal component, but is not tightly associated with small nuclear RNAs. It appears to associate with the spliceosome concomitant with or just after dissociation of the U4 small nuclear RNA during spliceosome assembly. We have found that PRP19 is associated with a protein complex in the splicing extract and that at least one of the associated components is essential for splicing. Taking advantage of the epitope tagging technique, we have isolated the PRP19-associated complex by affinity chromatography. The isolated complex is functional for complementation for the heat-inactivated prp19 mutant extract, and consists of at least seven polypeptides in addition to PRP19. At least three of these can interact directly with the PRP19 protein. We also show that the PRP19 protein itself is in an oligomeric form, which might be a prerequisite for its interaction with these proteins.

Yeast precursor mRNA processing protein PRP19 associates with the spliceosome concomitant with or just after dissociation of U4 small nuclear RNA.

During assembly of the spliceosome, the U4 small nuclear RNA (snRNA) interacts with the spliceosome as a preformed U4/U6-U5 triple small nuclear ribonucleoprotein (snRNP) complex. Subsequently, U4 becomes loosely associated with the spliceosome, whereas U5 and U6 remain tightly associated, suggesting unwinding of the U4/U6 duplex. We show that this step of the assembly process can be blocked by limiting the ATP concentration in the splicing reaction. We also show that the yeast precursor mRNA processing protein PRP19 becomes associated with the spliceosome during this transition. Thus, PRP19 may function in this step of spliceosome assembly.

PRP19: a novel spliceosomal component.

We have isolated the gene of a splicing factor, PRP19, by complementation of the temperature-sensitive growth defect of the prp19 mutant of Saccharomyces cerevisiae. The gene encodes a protein of 502 amino acid residues of molecular weight 56,500, with no homology to sequences in the data base. Unlike other PRP proteins or mammalian splicing factors, the sequence of PRP19 has no discernible motif. Immunoprecipitation studies showed that PRP19 is associated with the spliceosome during the splicing reaction. Although the exact function of PRP19 remains unknown, PRP19 appears to be distinct from the other PRP proteins or other spliceosomal components.

The yeast PRP19 protein is not tightly associated with small nuclear RNAs, but appears to associate with the spliceosome after binding of U2 to the pre-mRNA and prior to formation of the functional spliceosome.

We have previously shown that the yeast PRP19 protein is associated with the spliceosome during the splicing reaction by immunoprecipitation studies with anti-PRP19 antibody. We have extended such studies by using extracts depleted of specific splicing factors to investigate the step of the spliceosome assembly process that PRP19 is involved in. PRP19 was not associated with the splicing complexes formed in U2- or U6-depleted extracts but was associated with the splicing complex formed in heat-inactivated prp2 extracts. This finding indicates that PRP19 becomes associated with the splicing complexes after or concomitant with binding of the U6 small nuclear ribonucleoprotein particle (snRNP) to the precursor RNA and before formation of the functional spliceosome. We further analyzed whether PRP19 is an integral component of snRNPs. We have constructed a strain in which an epitope of nine amino acid residues recognized by a well-characterized monoclonal antibody, 12CA5, is linked to the carboxyl terminus of the wild-type PRP19 protein. Immunoprecipitation of the splicing extracts with anti-PRP19 antibody or precipitation of the extracts prepared from the epitope-tagged strain with the 12CA5 antibody did not precipitate significant amounts of snRNAs. Addition of micrococcal nuclease-treated extracts to the PRP19-depleted extract restored its splicing activity. These results indicate that PRP19 is not tightly associated with any of the snRNAs required for the splicing reaction. No non-snRNP protein factor has been demonstrated to participate in either step of the spliceosome assembly pathway that PRP19 might be involved in. Thus, PRP19 represents a novel splicing factor.