RBM25 localizes to splicing factor-rich nuclear speckles via the ER domain.
RBM25 consists of a proline-rich region and an RRM domain at the amino-terminal end, an RE/RD-rich (ER) domain in the central region, and a PWI domain at the carboxyl-terminal end (Fig. ). RBM25 is known to localize to nuclear speckles (16
). Our Ab against the C-terminal domain of RBM25 confirmed that endogenous RBM25 localized predominantly in the nuclear speckles as a punctate structure (Fig. , −DRB, RBM25). As previously reported, SC35 also localized to the nuclear speckles (Fig. , −DRB, SC35). Superimposition of RBM25 and SC35 revealed that both proteins are intensely colocalized in the same region (Fig. , −DRB, SC35 + RBM25). Upon treatment with DRB, an inhibitor of RNA polymerase II-dependent transcription, both proteins relocated to enlarged and more round nuclear speckles (Fig. , +DRB, SC35, and RBM25). Speckles function as storage compartments that can supply splicing factors to active transcription sites. Splicing factors are recruited from speckles to sites of transcription (30
); conversely, splicing factors accumulate in enlarged and rounded speckles when transcription is inhibited (30
). The colocalization and co-redistribution of RBM25 and SC35 suggest that RBM25 is organized in a pattern very similar to that of other splicing factors during states of transcriptional activity and quiescence.
FIG. 1. RBM25 localizes to splicing factor-rich nuclear speckles through the ER domain. (A) Schematic diagram of the structural organization of RBM25. RBM25 is composed of a proline-rich region (P) and an RRM at the amino-terminal end, an RE/RD-rich (ER) domain (more ...)
To further characterize the domain in RBM25 responsible for speckle localization, we expressed full-length RBM25, as well as its individual RRM, ER, and PWI domains, fused with EGFP in HeLa cells and analyzed its localization relative to splicing factor SC35. EGFP was detected throughout the cell when transfected with the vector alone (Fig. , GFP vector, GFP). The localization of full-length RBM25 was similar to that of endogenous RBM25; it colocalized with SC35 in nuclear speckles (Fig. , RBM25/FL, RBM25 + SC35). A different labeling pattern was observed for the RRM- and PWI-EGFP fusions. Both domains diffusely localized throughout the nucleus without any apparent accumulation on the speckles (Fig. , RBM25/RRM and RBM25/PWI, GFP). The ER domain produced a speckle pattern within the nucleus that coincided with that of SC35 (Fig. , RBM25/ER, RBM25 + SC35). Nuclear speckle localization of the ER domain was not cell type specific, as similar localization was seen in transfected murine erythroleukemia cells (data not shown). Thus, the ER domain of RBM25 is responsible for targeting RBM25 to the nuclear speckle.
RBM25 overexpression correlates with increased apoptotic cell death.
Although transient transfection of RBM25 localized to the nuclear speckles, our attempts to achieve stable RBM25 expression lines failed. These cells began to undergo apoptosis between 48 and 72 h after RBM25 introduction.
To gain more insight into the nature of these events, we first transfected HEK293 cells with EGFP or RBM25/EGFP and subsequently determined the extent of cell death by annexin V binding, cleavage of caspase 3, and nuclear fragmentation assays. Cells harvested 48 h after transfection showed a threefold increase in annexin V-positive cells in RBM25/EGFP-transfected cells compared with that of EGFP cells (Fig. ). Increased expression of RBM25 also significantly increased the percentage of activated-caspase 3-stained, GFP-positive populations (Fig. ). Furthermore, a significant fraction of RBM25-overexpressing cells displayed typical nuclear fragmentation associated with apoptotic cell death (Fig. ). These results suggest that an elevation in RBM25 expression caused an increase in apoptotic cell death, indicating that a correlation exists between RBM25 activity and induction of apoptosis.
FIG. 2. Increased RBM25 expression correlates with induction of apoptosis. HEK293 cells were transfected with EGFP or RBM25/EGFP. The development of apoptosis was assessed by annexin V binding of GFP-positive cells, by activated (cleaved) caspase 3 immunofluorescent (more ...)
We then documented the time course of this effect as revealed by nuclear fragmentation in approximately 500 RBM25/GFP-expreesing cells at 24, 48, and 72 h after transfection. At 24 h, different degrees of RBM25/GFP expression, as judged by the intensity/brightness of green fluorescence, were observed. In cells exhibiting “average” brightness, which applied to approximately 95% of the cells, RBM25/GFP localized to nuclear speckles. Of interest, nuclear fragmentation occurred in ~5% of the cells; these cells exhibited above-average brightness. The cells in Fig. expressed an average intensity of RBM25/GFP and nuclear speckle localization and are thus indicative of the vast majority of the cells observed.
At 48 h posttransfection, RBM25/GFP accumulated in large aggregates in the nuclei of approximately 25% of the expressing cells (Fig. , RBM25-GFP, GFP, arrow). These cells displayed various degrees of nuclear fragmentation (Fig. , RBM25-GFP, DAPI, arrow). The number of cells exhibiting nuclear fragmentation increased to 50% as transfected cells proceeded to 72 h. We were unable to select stable lines carrying RBM25 due to cell death caused by its overexpression.
We further analyzed the average exogenously expressed RBM25 in the transfected cells by Western blot analysis with an anti-RBM25 Ab. At 48 h posttransfection, an ~1.3- to 1.5-fold increase in RBM25/GFP expression (Fig. , RBM25-GFP, RBM25-GFP) over that of endogenous RBM25 (Fig. , RBM25-GFP, RBM25) was detected in total cell lysates. Taking into account the facts that every cell did not express equal amounts of RBM25/GFP and that only approximately 80% transfection efficiency was achieved, we estimate that the average RBM25/GFP expression is approximately 1.6- to 2.0-fold over that of endogenous RBM25. This amount of overexpression is sufficient to induce apoptosis.
Subcellular localization and expression of RBM25 during induced apoptosis.
To examine whether RBM25 expression and localization changed during apoptosis, HEK293 cells were treated with staurosporine for 0, 24, and 48 h and analyzed. Nuclear speckle localization of RBM25 was observed in untreated cells (Fig. , 0 h, RBM25). At 24 h, in the majority of the cells, RBM25 accumulated in large aggregates in the nucleus; these aggregates appeared to reside in subnuclear locations containing less condensed DNAs. Nuclear fragmentation was observed in a small population of cells. As apoptosis proceeded to 48 h, RBM25 accumulated in the subnuclear compartment with less condensed DNAs in some cells (Fig. , 48 h, RBM25, DAPI, merge, arrow) and a diminished RBM25 immunostaining signal was present in the majority of the cells with nuclear fragmentation (Fig. , 48 h, RBM25, DAPI, merge, arrowhead).
FIG. 3. Expression and localization of RBM25 during staurosporine-stimulated apoptosis in HEK293 cells. (A) Cellular localization of RBM25 at the indicated time points after staurosporine stimulation. HEK293 cells were mock treated or treated with 500 nM staurosporine (more ...)
We then examined whether RBM25 expression changed during the process. Equal amounts of cell lysates from both induced and uninduced cells were blotted with an anti-RBM25 Ab. An almost equally expressed 120-kDa band was detected in all samples (Fig. , RBM25, lanes 0, 24, 48, and 72). An additional higher-molecular-weight band was also detected in time zero and uninduced 24-h lysates (Fig. , RBM25, lanes 0 and −24). These results suggest that RBM25 expression changes could result from either posttranslational modification or expression of different isoforms. Western blot analysis also clearly demonstrated that the disappearance of RBM25 at 48 h in cells in which apoptosis had been induced was not due to protein degradation because RBM25 remained as intact and abundant in Western blot assays of apoptotic cells as it appeared at time zero (Fig. , lanes 0 and 48). These results imply a possible conformational change occurring in RBM25 that modifies the antigenic epitope recognized by the anti-RBM25 Ab.
RBM25 overexpression affects Bcl-x isoform expression.
The observations that RBM25 colocalized with splicing factors in the nuclear speckles and that its overexpression resulted in increased apoptotic cell death suggest a possible role for RBM25 in splicing regulation involved in the apoptotic pathway. This prompted us to search for targets of RBM25 with a focus on apoptotic factors. A large number of these factors are regulated via alternative splicing, a process that allows for the production of discrete protein isoforms with distinct apoptotic functions (42
We analyzed a panel of apoptotic factors, Mcl1 (1
), caspase-3 (22
), Bcl-x (4
), and Fas (13
), for the expression of their isoforms in response to RBM25 overexpression (Fig. ). Expression of RBM25 was validated by Western blotting with anti-HA Abs (Fig. , anti-HA). Among the tested factors, increased Bcl-xS
and reduced Bcl-xL
were noted in the presence of RBM25 (Fig. , Bcl-x), implying that RMB25 may regulate Bcl-x alternative splicing. Bcl-x pre-mRNA uses an alternative 5′ ss to produce the antiapoptotic Bcl-xL
or the proapoptotic Bcl-xS
isoform. RBM25 exerted its effect on more efficient utilization of the Bcl-xS
5′ ss, resulting in an increased ratio of Bcl-xS
. On the other hand, no effects on capase-3, Mcl1, and Fas were detected (Fig. , casp-3, Mcl1, and Fas). Since the inclusions of the alternative exons in these transcripts are already vastly predominant, their response to RBM25 was then further evaluated in an RBM25 knockdown background (Fig. ). Treatment of cells with an RBM25 shRNA depleted the endogenous RBM25 by 85% (Fig. , anti-RBM25, lanes non- and sh-82); nevertheless, no changes in the splicing pattern were detected. The alternative splicing of caspase-3, Mcl1, and Fas was not affected by RBM25 (Fig. , casp-3, Mcl1, and Fas). Thus, the effect of RBM25 on Bcl-x pre-mRNA processing was specific and not attributable to a generalized effect on the RNA splicing machinery.
FIG. 4. Effect of RBM25 on alternative splicing of selected apoptotic factors. pCDNA3.1-HA-RBM25-transfected or RBM25 shRNA-depleted HeLa cells were analyzed for alternative splicing patterns of exon 6 of caspase 3, exon 2 of Mcl1, Bcl-xS and Bcl-xL of Bcl-x, (more ...) Increased RBM25 expression correlates with increased Bcl-xS 5′ ss usage.
To investigate the regulation of Bcl-x splicing by RBM25, we used a Bcl-x minigene (a gift from C. E. Chalfant, Virginia Commonwealth University, Richmond) that spans the entire alternatively spliced region from exon 1 to exon 3, with a shortened intron 2 (Fig. ). The Bcl-x minigene produced the same Bcl-x splicing pattern as seen with endogenous Bcl-x and produced two splice variants of the Bcl-x transcripts with a ratio of Bcl-xS to Bcl-xL of 0.31 in HeLa cells (Fig. , lane 0). Cotransfection of pcDNA3.1-HA-RBM25 with this reporter activated splicing pathways which led to increased short variant Bcl-xS with respect to transfection of pcDNA3.1-HA alone (Fig. , lanes 0, 0.25, 0.5, 0.75, 1.00, and 1.50). The selection of the upstream 5′ ss in exon 2, resulting in the production of Bcl-xS, was stimulated in an RBM25 dose-dependent manner (Fig. ). The addition of 0.25 μg RBM25 increased the ratio of Bcl-xS to Bcl-xL to 0.64, while the ratio increased to 2.3 when 1.5 μg of RBM25 was introduced. The expression of RBM25 was validated by a Western blot assay with anti-HA Abs (Fig. , anti-HA). These results suggest that the Bcl-x reporter gene responded to RBM25 by enhancing Bcl-xS 5′ ss usage.
FIG. 5. RBM25 regulates Bcl-x 5′ ss selection. (A) Schematic diagram of the Bcl-x minigene spanning the entire alternatively spliced region from exon 1 to exon 3, with a shortened intron 2. Two splice variants derived from the Bcl-x gene, proapoptotic (more ...) Depletion of RBM25 reduces Bcl-xS 5′ ss usage.
The observation that increased expression of RBM25 could enhance the usage of the Bcl-xS 5′ ss prompted us to examine whether reduction of RBM25 expression would block the usage of the same 5′ ss. Several RBM25 shRNA constructs (Fig. ), individually or in combination, reduced endogenous RBM25 expression in HeLa cells (Fig. , RBM25, lanes sh81, sh82, sh83, sh84, and all). A nonfunctional control nonsilencing shRNA served as a control (Fig. , RBM25, lane non).
FIG. 6. Depletion of RBM25 by RNA interference leads to reduction of proapoptotic Bcl-xS. (A) Schematic of RBM25 regions targeted by RBM25 shRNAs (sh81, sh82, sh83, and sh84). P, proline-rich region; RRM, RNA recognition motif; ER, glutamic acid/arginine-rich (more ...)
We analyzed Bcl-x splicing patterns in RBM25 shRNA-treated cells. The nonsilencing control did not affect the splicing patterns (Fig. , lane non) compared with that of untreated cells (Fig. , lane 0). Reduction in RBM25 clearly affected the Bcl-x splicing pattern, in which a discernible decrease in Bcl-xS 5′ ss usage was observed in RBM25 shRNA-treated cells (Fig. , lanes non, sh81, sh82, sh83, sh84, and all). Bcl-xS production was reduced by 3- to 10-fold. The decrease corresponded to the potency of the sh-RBM25 used to knock down RBM25 (Fig. , expression, lanes non, sh81, sh82, sh83, sh84, and all). These results further suggest an involvement of RBM25 in the determination of 5′ ss selection in the Bcl-x gene.
RBM25 promotes Bcl-xS 5′ ss usage through CGGGCA in exon 2.
To examine whether RBM25 exerted its activity through an interaction with a cis-acting element, we first identified RNA sequences that have an effect on 5′ ss selection in response to RBM25 expression levels. We generated mutant forms of Bcl-x minigenes in which we replaced several sequences spanning exon 2 (Fig. ). Except for Mu1 and Mu5 (Fig. , −RBM25, lanes Mu1 and Mu5), the replacements affected the splicing pattern with either increased (Fig. , −RBM25, lane Mu2) or decreased ratios of Bcl-xS to Bcl-xL (Fig. , −RBM25, lanes Mu3 and Mu4) compared with that of the WT (Fig. , −RBM25, lane WT). These results suggest that the exonic sequences could activate or repress Bcl-xS 5′ ss usage.
FIG. 7. A sequence motif within exon 2 is important for regulated splicing of Bcl-x by RBM25. (A) A diagram indicating mutated sequences and nucleotides replaced in the Bcl-x minigene tested in the experiment. (B) Analysis of the effect of mutations on Bcl-x (more ...)
We then examined whether the mutated minigenes would still be responsive to RBM25 by cotransfection of RBM25 and the minigenes into HeLa cells. All but one responded to the stimulation of RBM25 by increasing the ratio of Bcl-xS to Bcl-xL (Fig. , +RBM25, lanes WT, Mu1, Mu2, Mu3, and Mu5). Mutation of an element, CGGGCA, located 64 to 69 nt upstream of the Bcl-xS 5′ ss drastically reduced Bcl-xS expression and shifted the ratio of Bcl-xS to Bcl-xL from 0.33 to 0.02 (Fig. , −RBM25, lane Mu4). Furthermore, mutation of this element abolished the ability of RBM25 to affect 5′ ss selection (Fig. , +RBM25, lane Mu4). These results suggest that RBM25 might exert its activity through its binding to the CGGGCA element.
RBM25 associates with Bcl-x transcript and interacts with CGGGCA in vitro.
We then investigated whether the endogenous Bcl-x RNA was associated with RBM25 by using an RIP assay with an anti-RBM25 Ab. RNA templates retrieved by RIP were analyzed by RT-PCR with primer sets to detect Bcl-x RNA. As shown in Fig. , Bcl-x RNA was detected in both input and anti-RBM25 precipitates (Fig. , input, α-RBM25, lanes +RT). The specificity of RNA IP is evidenced by the fact that IgG did not precipitate Bcl-x RNA (Fig. , Bcl-x, IgG, lane +RT). Furthermore, no products were detected in the absence of RT (Fig. , Bcl-x, α-RBM25, lane −RT). These results suggest that Bcl-x RNA associates with RBM25. To ensure that anti-RBM25 Ab did not pull down any nonspecific RNA sequences, we amplified isolated RNA for the presence of Mcl1 with its specific primer sets. While Mcl1 RNA was detected in the input, it was not found in anti-RBM25 precipitates (Fig. , Mcl1, input and α-RBM25, lanes +RT). These results are consistent with the notion that Mcl1 is not a substrate of RBM25 and validate the specificity of the RIP assay.
FIG. 8. RBM25 binds to Bcl-x RNA through the exonic sequence CGGGCA. (A) Bcl-x RNAs detected by RIP with an anti-RBM25 Ab. HeLa cells were fixed with 1% formaldehyde, and RIP was carried out with the cross-linked cell lysate and anti-RBM25 Ab or control (more ...)
To further examine whether RBM25 binds to CGGGCA directly, we performed an electrophoretic mobility shift analysis with CGGGCA-containing RNA and purified RBM25 protein. We examined the specific interaction between RBM25 and CGGGCA with a probe derived from the WT consisting of the sequences flanked by 6 nt upstream and 8 nt downstream (Fig. , WT). The mutant sequence TTAGAG, flanked by identical upstream and downstream sequences (Fig. , Mut), served as a control. The intensity of the retarded band increased when the WT probe was incubated with increasing amounts of RBM25 (Fig. , WT, lanes 0, 0.5, 1.0, 2.0, and 4.0 μM). GST alone did not bind to the RNAs (Fig. , WT, lane GST). No retarded bands were observed when increased amounts of RBM25 were incubated with the mutant probe (Fig. , Mut, lanes 0, 0.5, 1.0, 2.0, and 4.0 μM). To assess the specificity of this WT RNA-protein interaction, a binding competition assay was performed with a 1-, 5-, or 20-fold molar excess of unlabeled WT or mutant RNA. The RNA-protein complexes were partially competed away by a 5-fold excess, and completely competed away by a 20-fold excess, of WT (Fig. , WT) but not mutant RNA (Fig. , Mut), suggesting that the observed WT RNA-RBM25 interaction is CGGGCA sequence specific.
CGGGCA activates splicing from a weak 5′ ss in a test E1A reporter system.
To pinpoint whether CGGGCA conferred RBM25 sensitivity, we examined the element in the context of the E1A reporter gene. A naturally occurring CGGGCA element is located 88 to 94 nt upstream of the 13S 5′ ss in E1A (Fig. ). Transfection of an E1A minigene alone produced three major classes of mRNAs with ~46% 13S, 38% 12S, and 16% 9S through the use of alternative 5′ ss (Fig. , non, lane −RBM25). The 13S 5′ ss is much stronger than the 12S or 9S 5′ ss. Unexpectedly, the presence of cotransfected RBM25 did not change the overall splicing pattern (Fig. , non, lane +RBM25). We then asked whether the effect of CGGGCA would be more potent in the context of a weak 9S 5′ ss. We created E1A+WT and E1A+Mu4 constructs in which the WT (UGGGCA) or the Mu4 (TTAGAG) sequence was inserted at positions 64 to 69 upstream of the 9S 5′ ss, respectively. The presence of UGGGCA resulted in a shift in splicing favoring the use of the 9S 5′ ss; an elevated 9S accounted for ~25% and a concomitant reduction of 12S to ~30% (Fig. , WT, lane −RBM25). Cotransfection of RBM25 promoted ~25% more 9S splicing (Fig. , WT, lane +RBM25). The insertion of Mu4 sequence exerted no significant effect on splicing in either the absence or the presence of RBM25 (Fig. , Mu4, lanes −RBM25 and +RBM25). These results suggest that UGGGCA could enhance the utilization of the weak 9S 5′ ss but not that of a strong 13S 5′ ss through its interaction with RBM25.
FIG. 9. Effect of CGGGCA on E1A reporter gene 5′ ss selection in vivo. (A) Schematic diagram of the E1A minigene and its major splicing products. E1A+WT and E1A+Mu4 constructs were created in which the WT (UGGGCA) or the Mu4 (TTAGAG) sequence (more ...) RBM25 facilitates the recruitment of U1 snRNP to the weak 5′ ss.
The observation that CGGGCA exerted its effect on a weak 5′ ss prompted us to examine how RBM25 activated a 5′ ss with Bcl-x. We first analyzed the strength of Bcl-x 5′ ss with the Analyzer Splice Tool (http://ast.bioinfo.tau.ac.il/SpliceSiteFrame.htm
), which uses an algorithm based on the work of Shapiro and Senapathy (45
) to calculate the scores of donor and acceptor sequences. Bcl-xS
possesses a weak 5′ ss with a score of 72.66, while Bcl-xL
5′ ss is relatively stronger at 81.51; the reference consensus sequence CAG/GTAAGT has a score of 100 (Fig. ).
FIG. 10. RBM25 associates with hLuc7A and promotes U1 snRNP binding to a weak 5′ ss in a CGGGCA-dependent manner. (A) The WT, CGGGCA-to-TTAGAG mutant (Mu4), or consensus 5′ ss mutant (5′cons) Bcl-x minigene with the mutation sequence indicated (more ...)
Early recognition of the 5′ ss involves base-pairing interaction with the 5′ end of U1 snRNA. We performed psoralen-mediated UV cross-linking assays to examine whether U1 snRNP is recruited to transcripts containing the Bcl-xS 5′ ss (Fig. , WT). A psoralen-dependent cross-linked U1 snRNA/Bcl-x was detected upon incubation of a radioactively labeled WT sequences in HeLa cell nuclear extracts (Fig. , WT, lane +/−). This interaction was significantly enhanced when ~300 ng of purified RBM25 was added (Fig. , WT, lane +/+), suggesting that the presence of RBM25 facilitates U1 snRNP binding.
To determine whether the sequence CGGGCA is required for U1 interaction with the Bcl-xS 5′ ss, we repeated the psoralen cross-linking assay with a Mut4 mutant probe. Mutations in the RBM25 binding site reduced the cross-linking product to a small extent (Fig. , Mu4, lane +/−) compared with that of the WT probe (Fig. , WT, lane +/−). In contrast to the WT probe, the addition of RBM25 did not promote U1 cross-linking to the Mu4 transcript (Fig. , Mu4, lane +/+). These results suggest that RBM25-dependent U1 recruitment requires CGGGCA.
Data from Fig. suggest that the presence of CGGGCA, followed by a strong 13S 5′ ss, had no effect on 13S splicing activity in the presence of RBM25. We thus tested whether an increased strength in Bcl-xS 5′ ss would attenuate the effects of RBM25 in a cross-linking assay with Bcl-xS and a consensus 5′ ss probe (Fig. , 5′cons). The 5′cons transcripts cross-linked to U1 snRNA (Fig. , 5′cons, lane +/−) much more strongly than did the WT in the absence of added RBM25 (Fig. , WT, lane +/−). Addition of RBM25 did not increase 5′cons cross-linking to U1 snRNA (Fig. , 5′cons, lane +/+). These data suggest that the contribution of RBM25 to U1 snRNP recruitment is particularly critical for a weak 5′ ss.
Subsequently, we analyzed the effect of the strength of Bcl-xS 5′ ss on splicing and its response to RBM25 in HeLa cells. Strengthening Bcl-xS 5′ ss to consensus sequence led to ~100% usage of the 5′ ss in the absence of RBM25 (Fig. , 5′cons, lane −RBM25). Although RBM25 had an enhanced effect on the selection of the weak WT 5′ ss (Fig. , WT, lane +RBM25), a strong 5′ ss did not require facilitation by RBM25 for the 5′ ss selection (Fig. , 5′cons, lane +RBM25). These results suggest that RBM25 may facilitate WT 5′ ss recognition by improving the interaction between U1 snRNA and the weak 5′ ss.
RBM25 associates with hLuc7A.
Several positive regulators activate splicing of alternative exons with a weak 5′ ss by promoting interaction of U1 snRNP with the 5′ ss. U1 snRNP recognizes the 5′ ss and is among the first factors to interact with the pre-mRNA to form “complex E,” which commits the pre-mRNA to the splicing pathway (26
). We explored the possibility that RBM25 associates with the U1 snRNP complex and plays a role in recruiting U1 snRNP to the weak 5′ ss. U1 snRNP is composed of a 165-nt RNA (U1 snRNA), seven different Sm proteins common to other snRNP, and three U1-specific proteins (U1-70K, U1-A, and U1-C) (38
). To probe the interactions between RBM25 and U1 snRNP, RBM25 was immunoprecipitated from HeLa cell nuclear extracts and analyzed by Western blotting for the presence of U1-specific proteins. No U1-specific proteins were detected in the precipitates (data not shown).
Human Luc7A has recently been shown to associate with U1-A and U1 snRNA and stabilize pre-mRNA-U1 snRNP interaction (36
). We thus examined whether RBM25 associated with hLuc7A in the immunoprecipitates. Since RBM25 contains RRM and PWI motifs, both of which can bind to RNA, we also examined whether the association is RNA mediated. As shown in Fig. , hLuc7A was abundantly detected in anti-RBM25 immunoprecipitates in an RNase-independent manner (Fig. , α-RBM25, lane p, +RNase). The association of hLuc7A with RBM25 is most likely through the carboxyl half of hLuc7A because both the full length and the C half of hLuc7A, but not the N half, pulled down RBM25 in a GST pull-down experiment (Fig. ). Furthermore, hLuc7A colocalized in the nuclear speckles with RBM25 (Fig. , RBM25+Luc7A). These results suggest that binding of RBM25 to the CGGGCA sequence may help recruitment of U1 snRNP to the weak 5′ ss through its interaction with U1 snRNP-associated hLuc7A and provide a molecular mechanism for the function of this splicing regulator.