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Mol Cell Biol. Oct 2008; 28(19): 6033–6043.
Published online Jul 21, 2008. doi:  10.1128/MCB.00726-08
PMCID: PMC2547008

Multiple and Specific mRNA Processing Targets for the Major Human hnRNP Proteins[down-pointing small open triangle]

Abstract

Alternative splicing is a key mechanism regulating gene expression, and it is often used to produce antagonistic activities particularly in apoptotic genes. Heterogeneous nuclear ribonucleoparticle (hnRNP) proteins form a family of RNA-binding proteins that coat nascent pre-mRNAs. Many but not all major hnRNP proteins have been shown to participate in splicing control. The range and specificity of hnRNP protein action remain poorly documented, even for those affecting splice site selection. We used RNA interference and a reverse transcription-PCR screening platform to examine the implications of 14 of the major hnRNP proteins in the splicing of 56 alternative splicing events in apoptotic genes. Out of this total of 784 alternative splicing reactions tested in three human cell lines, 31 responded similarly to a knockdown in at least two different cell lines. On the other hand, the impact of other hnRNP knockdowns was cell line specific. The broadest effects were obtained with hnRNP K and C, two proteins whose role in alternative splicing had not previously been firmly established. Different hnRNP proteins affected distinct sets of targets with little overlap even between closely related hnRNP proteins. Overall, our study highlights the potential contribution of all of these major hnRNP proteins in alternative splicing control and shows that the targets for individual hnRNP proteins can vary in different cellular contexts.

Alternative splicing is a critical process that ensures the production of a multitude of proteins from a limited set of mammalian genes (7, 41). Alternative splicing decisions are regulated by a large collection of RNA-binding proteins (RBPs) that bind to pre-mRNAs in the nucleus (5). The heterogeneous nuclear ribonucleoparticle (hnRNP) proteins are among the most abundant of such proteins, and more than 20 of them have been characterized and given alphabetical names based on size from hnRNP A1 to hnRNP U (17). These proteins have been implicated in a variety of biological processes including telomere biogenesis, translation, and RNA stability, and several (e.g., hnRNP A1, A2, F, H, I [PTB], G, and L) have documented roles in splicing (34, 38). hnRNP A1 has been implicated in the splicing control of many genes, including the A1 gene itself, the caspase-2 gene, c-src, and the SMN2 gene (12), and several exons of human immunodeficiency virus type 1; the very similar hnRNP A2 protein (68% identity) appears to display comparable activity (4, 10, 29, 42). While the related hnRNP F and H proteins play a role in splicing control of many genes, including c-src, Bcl-x, cystathionine β-synthase, and several HIV alternative exons (38), it is unclear whether F and H have completely redundant activities. hnRNP I (PTB) is another well-known splicing regulator that has been mostly associated with splicing repression (54, 59). A recent global analysis of PTB and its neural paralogue nPTB has revealed their role in the control of murine neuron-specific splicing (8). hnRNP G (RBM-X) has been implicated in the splicing control of several pre-mRNAs including SMN and βs-tropomyosin (38, 45). The human hnRNP L protein promotes exon inclusion in the eNOS pre-mRNA (27) and exclusion of the variable exon in CD45 (24). A recent microarray screen has identified several novel pre-mRNA targets of hnRNP L (28).

A role in splicing for the other major hnRNP proteins is less conclusive, mostly because the number of examples remains limited. A function for hnRNP C in splicing is controversial since the initial reports were never confirmed (13, 52). hnRNP K has been associated with splicing efficiency (18) and also with one case of alternative splicing control in vivo using a minigene (56). Overexpression of hnRNP M can promote both exon inclusion and skipping events in a few alternative exons in 293T cells (25). hnRNP Q (Syncrip) is required for efficient pre-mRNA splicing in vitro (44), but the function of the very similar hnRNP R protein is unknown. It is unclear whether the mostly cytoplasmic hnRNP D protein (AUF1) and the related DL protein can modulate splicing.

To begin to assess the range of action of the major hnRNP proteins that function in mRNA processing and to explore the potential function of the others, we have analyzed the individual capacities of several of the major hnRNP proteins to affect the production of splice isoforms. We have selected alternative events that belong to a subset of apoptotic genes because the functional consequences of alternative splicing on apoptosis have been amply demonstrated (51). Indeed, the complex and delicate balance between the activities of pro- and antiapoptotic variants produced by APAF-1, Bcl-x, Fas, and caspases is often controlled through alternative splicing, and a number of studies have documented the contribution of hnRNP and hnRNP-like proteins in the control of splice site selection in apoptotic genes. For example, PTB antagonizes U2AF binding to the upstream 3′ splice site of Fas exon 6 by repressing exon definition (31). A role for PTB and hnRNP A1 has also been observed in the control of caspase-2 pre-mRNA splicing (14). Likewise, hnRNP F/H, along with the RBPs ASF/SF2, and Sam68 have been implicated in the control of Bcl-x splicing to produce pro- and antiapoptotic isoforms (21, 40, 48).

The first global study linking RBPs to alternative splicing was carried out by examining the impact of downregulating hundreds of RBPs on three genes in the Drosophila S2 cell line (47). A subsequent study reported the effect of depleting four RBPs on the alternative splicing of thousands of genes in the same cell line (6). In mammals, RNA interference assays were also used to study global effects of the hnRNP-like protein Nova2 in the mouse neocortex, of PTB in a mouse neuronal cell line, and of hnRNP L in HeLa cells (8, 28, 57).

We have combined an RNA interference approach with our high-throughput reverse transcription-PCR (RT-PCR) screening platform (32) to analyze the impact of downregulating 14 hnRNP proteins on 56 alternative splicing events (ASEs) in apoptotic genes expressed in the human cell lines HeLa, PC-3, and BJT. We find that all hnRNP proteins tested can affect the ratio of some splice isoforms in human cells. The impact of the individual knockdowns varies considerably, ranging from <2% of ASEs being affected by hnRNP M to >40% of all events affected by hnRNP K. Although many hnRNP proteins have the capacity to control the same splicing events in two or three cell lines, the contribution of several of these abundant proteins varies considerably in different cellular environments.

MATERIALS AND METHODS

Cell lines and siRNAs.

The cervical cancer cell line HeLa, the prostate cancer cell line PC-3, and an immortalized version of the normal foreskin BJ-TIELF (BJT) fibroblast cell line used in the present study have been described previously (49). HeLa, PC-3, and BJT cells were, respectively, grown in Dulbecco modified Eagle medium, Ham F-12 medium, and alpha-minimal essential medium supplemented with 10% fetal bovine serum. The siRNAs used to knockdown hnRNP protein expression were purchased from IDT (Coralville, IA), and their sequences are listed in Table S1 in the supplemental material. siRNAs were transfected into cells at a concentration of 100 nM using Lipofectamine 2000 (Invitrogen). Proteins and RNA were extracted from mock-transfected and small interfering RNA (siRNA)-transfected cells 96 h posttransfection.

Western analysis and quantitative real-time RT-PCR.

We used antibodies to assess the success of knockdown experiments. The same blot was decorated with an anti-actin antibody (Sigma A2066) to correct for total protein content in different lanes. Anti-hnRNP A1 and A2 antibodies and anti-hnRNP F and H antibodies were described previously (49, 50). Anti-hnRNP C1/C2 and G (C-17) were from Santa Cruz Biotechnologies, Inc. (sc-10037 and sc-4583, respectively); anti-hnRNP D/AUF1 was purchased from Upstate Biotechnology, Inc. (catalog no. 07-260); anti-JKTBP (hnRNP DL) was kindly provided by Michiyuki Yamada (Yokohama City University, Yokohama, Japan); anti-hnRNP I (PTB) was kindly provided by Doug Black (University of California at Los Angeles); anti-hnRNP K was kindly provided by Karol Bomsztyk (University of Washington); anti-hnRNP L was purchased from Abcam (ab6106); anti-hnRNP M was purchased from Abnova (H00004670-A01); and anti-Syncrip (hnRNP Q) was kindly provided by Mike Mueckler (Washington University).

When antibodies were not available or the quality of the Western analysis was unsatisfactory, we conducted quantitative real-time RT-PCR assays using Sybr green (Power Sybr green master mix, 2×; ABI catalog no. 4367660) to assess transcript levels. Aldolase A (RTPrimerDB ID: 915) was used as the housekeeping gene on the same samples. A total of 200 ng of RNA measured for integrity (using the Agilent Lab-on-Chip station) and quantification (using the Thermo Scientific NanoDrop) was reverse transcribed using random hexamers (Roche catalog no. 11034731001) with Transcriptor reverse transcriptase (Roche catalog no. 03531317) in a final volume of 10 μl. Then, 10 ng of cDNA was used for quantification in the presence of the specific primers at 0.2 μM in a 10-μl reaction performed in triplicate. For the list of genes, oligonucleotides, and expected sizes of qPCR products, see Table S2 in the supplemental material. Reactions were carried out in an ABI 7500 qPCR (Applied Biosystems, Foster City, CA) or Eppendorf Realplex. A first cycle of 10 min at 95°C was followed by 40 cycles of 15 s at 94°C, 20 s at 55°C, and 20 s at 68°C.

RT-PCR assays.

A set of 56 alternative splicing units from human apoptotic genes was selected from the AceView database. Sets of primers mapping in the exons flanking the ASEs were designed by using Primer3 with default parameters. For the global analysis of splice isoforms from apoptotic genes, total RNA was extracted using TRIzol and quantitated by using the Lab-on-Chip station (Agilent, Inc., Santa Clara, CA). A total of 2 μg of RNA was reverse transcribed using a mix of random hexamers and oligo(dT) and the Omniscript reverse transcriptase (Qiagen, Germantown, MD) in a final volume of 20 μl. Then, 20 ng of cDNA was amplified with 0.2 U/10 μl of HotStarTaq DNA polymerase (Qiagen) in the buffer provided by the manufacturer, without extra MgCl2 and in the presence of the specific primers (IDT) for each splicing unit (at concentrations ranging from 0.3 to 0.6 μM) and deoxynucleoside triphosphates. The list of units, oligonucleotides, and expected sizes of the RT-PCR products are shown in Table S3 in the supplemental material. Reactions were carried out in the GeneAmp PCR system 9700 (Applied Biosystems). A first cycle of 15 min at 95°C was followed by 35 cycles of 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C. The reaction was ended with the extension step of 10 min at 72°C. Visualization and analysis of amplified products was done by using the LabChip HT DNA assay on an automated microfluidic station (Caliper, Hopkinton, MA) (32).

Bioinformatic analysis.

The search for hnRNP binding motifs in cassette exons or single alternative 5′ or 3′ splice sites also included 150 nucleotides of upstream and downstream sequences but did not include sequences where the flanking introns were smaller than 150 nucleotides. The clustering was done by R using the R Project for Statistical Computing (http://www.r-project.org/) with the Euclidean distance metric.

RESULTS

We carried out a loss-of-function study in three cell lines: the cervical cancer HeLa cell line, the prostate cancer PC-3 cell line, and the BJT cell line (hTERT-immortalized foreskin fibroblasts derived from the BJ cell line) (49). Fourteen hnRNP proteins (A1, A2, C, D, DL, F, G [RBM-X], H, I [PTB], K, L, M, Q [Syncrip], and R) were each targeted with two distinct and nonoverlapping siRNAs. The combined knockdown of A1 and A2 was also performed. For the related hnRNP F and H proteins, we used one siRNA for each and a different siRNA targeting both F and H. Depletion efficiencies were evaluated 96 h posttransfection by Western blotting and/or quantitative RT-PCR assays (Table (Table11 and Fig. S1 in the supplemental material). All assays indicated that depletion had been achieved, except for hnRNP H and hnRNP Q in BJT cells, which remained refractory to RNA interference-mediated silencing even after repeated attempts.

TABLE 1.
Knockdown efficiencies

A set of 56 ASEs covering a variety of apoptotic genes was evaluated to assess the impact of individual hnRNP protein knockdowns. Primers were designed to cover alternatively spliced regions such that two distinct splice products of sizes ranging between 100 and 700 bp could be amplified. The relative abundance and size of each amplification product was estimated by capillary microfluidic fractionation using Caliper reading stations. For each reaction, the relative abundance of the products was converted to a percent splicing index Ψ, defined as the molarity of the long product divided by the combined molarities of the long and short products. A ΔΨ value, defined as Ψknockdown − Ψcontrol, was then computed. ΔΨ is therefore a measure of the relative shift in splice site selection compared to the control transfection performed on the same day. ΔΨ values were calculated for each ASE in each cell line that was treated with a given siRNA. A ΔΨ with a |Z|-score of 1.5 (1.5 standard deviations above or below the average variation observed for all controls performed with all splicing units in this cell line) was considered significant (a “hit”) only if it was detected with both siRNAs. Using two siRNAs for each hnRNP protein is important because the correlation scores for pairs of siRNAs with similar knockdown efficiencies varied greatly, indicating that some siRNAs can generate many false positives.

From the nearly 5,000 ΔΨ values collected, 107 splicing events had ΔΨ values with |Z|-scores above 1.5 with both siRNAs (Fig. (Fig.1).1). More than one-third of these hits resulted from knocking down hnRNP K and C. To test the reproducibility of our hit assignment, we repeated the entire knockdown sets for hnRNP K and C. As seen in Fig. S2 and S3 in the supplemental material, good agreement was observed between the initial and the corroborative sets (sensitivity, 55%; specificity, 95%). Eighty-two percent of all shifts and nonhits were confirmed.

FIG. 1.
Alternative splicing of 56 ASEs in response to knockdown of 14 hnRNPs with two siRNAs. Individual hnRNP knockdowns and ASEs are shown to indicate which knockdowns caused a shift in alternative splicing in various cell lines (B = BJT, H = ...

Impact of the individual knockdowns.

The profile for each individually targeted hnRNP protein is shown in Fig. Fig.1.1. For the purposes of the discussion below, we have combined the presentation of the hits for hnRNP A1 and A2, as well as for hnRNP F and H, because these proteins are structurally very similar and are considered functional homologues. HnRNP D and DL, as well as hnRNP Q and R, are also combined for structural reasons.

hnRNP A1 and A2.

The number of alternative splicing units controlled by A1 and A2 averaged 5% in HeLa and PC-3 cells for each protein. No hits were obtained in BJT cells, presumably because the knockdowns were less efficient (Table (Table1).1). Five of the six A1 hits were obtained when we knocked down both A1 and A2, but no new hits were observed. A1 and A2 did not share common hits, indicating distinct target specificity. This is a surprising observation because A1 and A2 are considered functional homologues, although this conclusion is based on in vitro studies (4, 10, 29, 42). The fact that only one of the five A2 targets shifted with a dual knockdown is similarly intriguing. It may be explained if downregulating A2 upregulates the expression of A1 and vice versa (49; data not shown). If A1 is the dominant splicing regulator, knocking down A1 would increase the expression of A2, but the majority of the hits would be caused by the A1 decrease. In contrast, knocking down A2 would increase A1 expression to reveal distinctly sensitive units. Consistent with this view, we have observed in the data set with a lower |Z|-score two cases where the knockdowns of A1 and A2 shift splicing in opposite directions (data not shown).

Knocking down hnRNP A1 and A2 promoted a mixture of exon exclusion and exon inclusion events, a finding consistent with the recognized behavior of these proteins in splice site selection (39).

hnRNP C.

Given the controversial role of hnRNP C in splicing, it was surprising to note that a drop in hnRNP C expression affected 30% of the ASEs, nearly half of them occurring in at least two different cell lines. Moreover, knocking down hnRNP C only promoted exon skipping, suggesting that hnRNP C acts as a splicing enhancer and enforces exon inclusion. However, knocking down hnRNP C also promotes the use of a more internal 3′ splice site on LRDD when the Z-score is lowered. Such a result can be explained if hnRNP C enhances the downstream 3′ splice site or, alternatively, if it represses the upstream site. More work will be required to ascertain the mechanism(s) by which hnRNP C affects splice site selection. Given the association of hnRNP C with the SWI/SNF chromatin remodeling complex (37) and the role of other SWI/SNF components in alternative splicing (2, 3), hnRNP C may mediate these effects cotranscriptionally. Among the hits seen in three cell lines, the depletion of hnRNP C promoted the skipping of an alternative exon of 129 nucleotides in the poly(rC) binding protein 4 (PCBP4; Fig. Fig.2).2). The corresponding protein lacks 43 amino acids just downstream of its first KH domain. Exon exclusion in BCL2L12 is also observed upon hnRNP C depletion (Fig. (Fig.2),2), a situation that causes a frameshift and consequent deletion of the downstream BH2 domain.

FIG. 2.
Examples of hnRNP-regulated ASEs (hits). Selected units that were affected by hnRNP C (PCBP4 and BCL2L12) and hnRNP K (APAF1 and PTK2B) knockdowns in all cell lines are presented. at the top is a diagram of the alternative splicing unit being monitored. ...

hnRNP D and DL.

Knocking down hnRNP D and DL had an impact on the ratio of a few isoforms, and there was no overlap in hits. Given that hnRNP D and DL are mostly known as cytoplasmic proteins involved in controlling mRNA stability, it is possible that the changes that we observed are caused by differences in the stability of the specific mRNA splice isoforms. Nonetheless, nuclear functions for hnRNP D have also been proposed (16, 30) and a direct impact on alternative splicing remains possible but will need to be confirmed in future experiments.

hnRNP F and H.

Three of the eleven hnRNP F and ten hnRNP H hits were common, indicating that these proteins are regulating different but overlapping sets of ASEs. Interestingly, 80% of the events caused by downregulating F and H involved exon skipping, indicating a prevalent role for these proteins in enforcing the inclusion of alternative exons, a finding consistent with their proposed role in intron definition (39). Intriguingly, knocking down hnRNP F in PC-3 cells promoted the inclusion of a cassette exon in PCBP4, while the same treatment promoted its exclusion in BJT cells.

hnRNP G (RBM-X).

Knocking down hnRNP G affected the splicing of seven ASEs, and most hits were cell line specific. Both exon inclusion and exon skipping events were associated with the downregulation of hnRNP G. Notably, we observed that the depletion of hnRNP G promoted exon 8 skipping in MAPT (also known as Tau) in PC-3 cells. A role for hnRNP G in Tau splicing was previously noted when overexpressing it in COS cells promoted Tau exon 10 skipping (60).

hnRNP I (PTB).

Six ASEs were sensitive to the knockdown of PTB. Half of them changed in at least two cell lines, making PTB a broad effector like hnRNP C and hnRNP K (see below). Downregulating PTB enhanced exon inclusion in four ASEs, which is consistent with the prototypical inhibitory function of PTB (54). In one case, the depletion promoted the inclusion of the penultimate exon of protein phosphatase 3 (PPP3CB) in all three cell lines. In contrast, two cases of improved exon skipping were observed, suggesting that PTB does not always act as a repressor (46). A decrease in exon inclusion promoted by PTB depletion was also recently observed in mouse cell lines (8). The same study examined the impact of knocking down PTB on the alternative splicing of approximately 1,300 exons and showed that 25% of neuronal splicing in mouse is regulated by PTB and nPTB. In N2A cells, PTB affected 5% of the global ASEs monitored. We had a slightly superior hit frequency for human PTB (10%), and half of these hits were seen in all three cell lines.

hnRNP K.

Few natural ASEs have been associated with hnRNP K function. We show here that nearly 50% of ASEs are affected when hnRNP K is depleted. Moreover, most hits were detected in at least two cell lines, and 25% of the hits occurred in all three cell lines, making hnRNP K the most general effector in our set of hnRNP proteins. The knockdown of Nova2, a member of the same family of KH domain-containing proteins affected 7% of the neuronal ASEs (57). As was observed for Nova2, downregulating hnRNP K promoted a mixture of exon skipping and exon inclusion events, establishing hnRNP K as a bidirectional alternative splicing effector.

The splicing changes elicited by the depletion of hnRNP K affected important domains of apoptotic proteins. For example, downregulating hnRNP K caused the skipping of a 129-nucleotide exon in apoptotic peptidase activating factor 1 (APAF1) (Fig. (Fig.2).2). The corresponding 43 amino acid residues are located in the first C-terminal WD40 domain of this protein. Depletion of hnRNP K also promoted the inclusion of 44 amino acids just upstream of the C-terminal caspase recruitment domain of the NLR family, pyrin domain-containing 1 (NALP1). hnRNP K knockdown caused the exclusion of a 126-nucleotide exon (Fig. (Fig.2)2) encoding 42 amino acids between the tyrosine kinase domain and the focal adhesion targeting region of protein tyrosine kinase 2 (PTK2B). Secreted phosphoprotein 1 (SPP1; also known as osteopontin) had 14 residues excluded with both hnRNP K siRNAs in all cell lines. This splice variant is known as osteopontin-b and has no known function. However, an adjacent splice variant (osteopontin-c) is implicated in breast and liver cancer (43). The breast cancer-associated protein C11orf17 (BCA3) is a protein kinase A-interacting protein, and the splice variant lacking the phosphorylation sites for various kinases is increased when hnRNP K is depleted.

hnRNP L.

Downregulating hnRNP L affected seven ASEs. Its depletion promoted six cases of exon skipping and one case of exon inclusion, confirming the dual ability for hnRNP L to promote both exon skipping and exon inclusion events (24, 26-28). We found that BCL2L12 was a target in all three cell lines, whereas PPP3CB was affected in both HeLa and PC-3 cells. In a recent genomewide array analysis, Bindereif and coworkers (28) used the Affymetrix GeneChip Exon 1.0 Array to find and validate 11 targets of hnRNP L in HeLa cells; none of those ASEs was in our inquiry set.

hnRNP M.

hnRNP M has been implicated in the splicing control of a few genes (25). Here we observed that downregulation of hnRNP M with two different siRNAs affected the alternative splicing of only one gene, APLP2. Induced inclusion was observed in all three cell lines. Interestingly, this alternative exon of APLP2 is also included in the MCF-7 breast cancer cell line and excluded in the MDA231 breast cancer cells relative to normal HMEC mammary cells (36).

hnRNP Q and R.

Although hnRNP Q and R are considered structurally very similar, they did not target common ASEs. All hits (four for Q and three for R) were cell line specific and while knocking down hnRNP Q only promoted the skipping of cassette exons, downregulating hnRNP R increased exon inclusion in one case.

Generic and cell line-specific effects of hnRNP protein knockdowns.

Although the depletion of hnRNP C, H, and Q only promoted exon skipping events, all other knockdowns produced a mixture of exon skipping and inclusion events. Overall, downregulating the expression of hnRNP proteins promoted slightly more exon skipping events than exon inclusion events, further confirming that hnRNP proteins are not exclusively involved in exon repression. In only one case did the knockdown of an hnRNP protein have a different outcome in different cell lines; depleting hnRNP F in PC-3 cells promoted the inclusion of a cassette exon in PCBP4, while the same procedure promoted its exclusion in BJT cells. Except for the fact that all four exon skipping events promoted by the depletion of hnRNP G occurred in PC-3 cells, there was no apparent cell line bias toward exon inclusion or skipping.

The total numbers of hits were comparable in HeLa and PC-3 cells, but the total number was approximately half in BJT cells (Fig. (Fig.3,3, total), most likely because of the smaller magnitude of some of the depletions achieved in the BJT cell line (Table (Table1),1), and the fact that the initial level of many hnRNP proteins (A2, C, DL, F/H, G, K, L, and R) was generally lower in BJT relative to the other cell lines (see Fig. S1 in the supplemental material). Out of nearly 5,000 reactions (56 ASEs in three cell lines knocked down for 14 hnRNP proteins with two siRNAs), our analysis revealed 10 ASEs that were controlled consistently by an hnRNP protein in all three cell lines (solid yellow boxes in Fig. Fig.1),1), with 21 additional cases occurring in two cell lines. We noted 74 cases where an hnRNP knockdown produced a hit in only one cell line. For five of them, the assessment in other cell lines was compromised by the fact that the ASE was already fully spliced in the direction of the shift before treating with siRNAs. Twenty-one cases could be considered significant hits in all three cell lines but with a |Z|-score inferior to 1.5 (indicated as dashed yellow boxes in Fig. Fig.1).1). We identified 22 cases of clear cell line-specificity of hnRNP protein action (see Table S4 in the supplemental material). The vast majority of these examples of cell line specificity could not be explained simply by a superior hnRNP protein knockdown efficiency. Further investigations will be required to establish whether cell line specificity is due to a required threshold of hnRNP protein expression, to the existence of variant forms of hnRNP proteins, or to variations in the combinatorial nature of splicing regulation.

FIG. 3.
Representation of hits for each hnRNP protein in the three cell lines. Venn diagrams are used to indicate the number of significant alternative splicing units for each hnRNP (ASEs with shift |Z|-scores >1.5 with both siRNAs) that ...

Although PC-3 and HeLa cells shared ca. 40% of their hits, common hits were more frequent with hnRNP K, C, and PTB (55% on average) than for other hnRNP proteins (22%). Thus, the range of action of different hnRNP proteins varied considerably.

Networks of regulation.

To assess the existence of regulatory networks involving ASEs and hnRNP proteins, we produced a cluster map (Fig. (Fig.4).4). The clustering analysis confirms that hnRNP F and H had similar action profiles. Overall, however, there was little target overlap between various hnRNP proteins. Even for the hnRNP proteins associated with most ASEs (K, C, and F+H), the hit overlap was not significantly above the frequency expected to occur by chance, suggesting that specific sets of hnRNP proteins do not systematically act in concert to control alternative splicing but rather have specific and nonredundant roles.

FIG. 4.
Clustered binary map of significantly shifting alternative splicing units. Binary ΔΨ data for the hnRNP knockdown affecting different ASEs in various cell lines. The cell line in which the shift was observed for a specific ASE is indicated ...

A few genes in our selected set contained two distinct alternative splicing units (C11orf17, CAPN3, NALP1, OPA1, and TNFRSF10Bb). In PC-3 cells, hnRNP K affected the alternative splicing of both ASEs of NALP1, while hnRNP F did the same for TNFRSF10B. In addition, hnRNP L influenced the splicing of both units of CAPN3 in HeLa cells. Thus, a specific hnRNP protein can coordinate the splicing of distinct exons in one gene. These results extend a previous observation indicating that the alternative splicing of mouse ASEs belonging to the same genes are often coordinated in a tissue-specific manner (19).

Figure S4 in the supplemental material combines the data in Fig. Fig.11 to show which splicing events were sensitive to which hnRNP proteins, regardless of cell line. Fourteen ASEs did not react to any of the knockdowns, and nineteen were affected by only one hnRNP protein. At the other end of the spectrum, a few units were sensitive to the downregulation of several hnRNP proteins. Nearly one-third of all units reacted to the downregulation of at least three different hnRNP proteins. In PC-3 cells, PPP3CB splicing was affected by hnRNP A1, K, L, and PTB; CAPN3b by hnRNP K, DL, G, and C; and BCL2L12 by hnRNP H, Q, L, G, and C. These results indicate that an intricate and often cell line-specific network of hnRNP proteins is dedicated to the control of individual ASEs.

A few ASEs were controlled by two or more common hnRNP proteins. FGFR10P in PC-3 cells and CAPN3b in HeLa cells were regulated by hnRNP K, F/H, and A2. In PC-3 cells, hnRNP Q, H, L, and G all affected both BCL2L12 and MAPT. This last example may point to the existence of networks of coregulation where the combinatorial action of several different hnRNP proteins is converging to coordinate the processing of a group of genes. However, in examining the positions of genes in the apoptotic pathway we could not discern biases in favor of specific portions of the pathway controlled by individual or groups of hnRNP proteins (data not shown but see Fig. S5 in the supplemental material for the distribution of hnRNP K and C hits in the apoptotic pathway). Thus, hnRNP proteins act on a variety of genes involved at various steps of the apoptotic pathway. Likewise, there was no indication that hnRNP proteins individually or collectively were dedicated to the control of only pro- or only antiapoptotic events or proteins.

Our RT-PCR approach validated the control of 19 ASEs that were not included as RefSeq events (see Table S5 in the supplemental material). Many of the splicing events controlled by hnRNP proteins are presumed to play a critical role in protein function because the events implicate residues directly located in functional protein domains (14 cases), change the reading frame (8 cases), or create a premature stop (2 cases) (see Fig. S6 and Table S5 in the supplemental material).

Binding motifs for hnRNP proteins in target genes.

We have searched ASEs for known binding motifs of hnRNP proteins (Table (Table2),2), restricting our search to the 49 cases of alternative splicing involving cassette exons or single alternative 5′ or 3′ splice sites. The A1 binding motif UAGG (1, 15) was found in all four A2 hits and one-third of the A1 hits. The A1 binding motif UAGAGU resembling the SELEX winner A1 binding site (9, 29) was found in two A2 hits and more rarely in nonhits (5 of 45), with a P value for motif enrichment of 0.086. Although no perfect match to the known six-nucleotide consensus binding sites (e.g., UAGGGU and UAGAGU) was found in A1 hits, the submotif GGGU occurred in five of the six A1 hits and all four A2 hits.

TABLE 2.
Prevalence of binding motifs for hnRNP proteins in ASEs

Only 5 of the 15 hnRNP C hits contained the motif UUUUU (22). Both hnRNP D motifs AUUUA and UUAG (35, 62) were found in one of the two hits. For hnRNP F and H, nearly all 29 hits had at least one GGGG binding motif (11). Examples of the distribution of these motifs in hits are shown in Fig. S7a in the supplemental material. GGGG and GGG motifs were significantly more represented in the F hits than in the nonhits (P = 0.02 and 0.004, respectively), while this was the case only for the GGG motif in the H hits (P = 0.03).

The hnRNP G binding motif AAGU (45) was found in five of the six hits. At least one of the PTB-binding motifs UCUU and CUCUCU was found in every PTB hit, with a slight enrichment for the CUCUCU motif (P = 0.06). Half of the units reactive to a depletion of hnRNP K contained the optimal binding motifs AUC3/4(A/U)2 (55). Although the enrichment for this motif in hits relative to nonhits was modest (P = 0.06), it became more significant (P = 0.02) when only cases where K depletion increased exon inclusion were considered. This enrichment of AUC3/4(A/U)2 sites upstream of included exons implicates hnRNP K in the suppression of 3′ splice site recognition and consequent inhibition of alternative exons (see Fig. S7b in the supplemental material). Finally, the depletion in hnRNP L revealed diversely positioned binding motifs (ACAC, CACA, TACA, ACAT, and CACC) but no overrepresentation relative to nonhits (see Fig. S7c in the supplemental material).

The presence of binding motifs in the vicinity of alternative splice sites supports a direct role for the cognate hnRNP protein in splice site selection. Although this was the case for the majority of the hits, there may be several reasons why not all hits contained the expected binding sites. First, the sequences of all possible binding sites may differ from the binding motifs that we have selected. Indeed, hnRNP proteins are notorious for their relaxed binding specificity (53). Second, binding sites may act from a greater distance than the 150-nucleotide limit that we imposed on our search. Elements controlling specific splicing decisions can be located at a considerable distance from the splicing event that they control (20, 33). Third, hnRNP proteins may be components of a larger complex whose overall binding selectivity may be dictated primarily by other RBPs in the complex. Fourth, changing the expression of one hnRNP protein may affect the expression of other RBPs that in turn control splicing decisions. To assess this possibility, we carried out quantitative RT-PCR assays to measure the transcript levels of 14 hnRNP genes when hnRNP C or K were knocked down in PC-3 cells (see Fig. S8 in the supplemental material). Depleting hnRNP C affected the expression of hnRNP D, DL, G, H, M, and R, but levels did not drop below 50% with both siRNAs. Only two of the seven hnRNP C hits were detected in the other knockdowns (OPA1b and BCL2L12 with hnRNP H). Depleting hnRNP K affected the expression of hnRNP A2, D, DL, G, H, L, M, and R, with the expression of D and DL falling below the 50% level. Of the 15 hnRNP K hits in PC-3 cells, NALP1b, PTK2B, CASP1, and FGFR10P were also hits with hnRNP H, and CASP1 was a hit with hnRNP A2. On the other hand, one hit in common with hnRNP H (OPA1b) and two hits in common with hnRNP DL (BARD1 and CAPN3b) occurred in the opposite direction. Thus, reducing the expression of specific hnRNP proteins can affect the expression of other hnRNP genes. Whether all of these changes in mRNA expression were accompanied by similar changes in hnRNP protein expression remains to be examined. We could not detect significant changes in the levels of hnRNP A1/A2 and F/H proteins in hnRNP K knockdowns, probably because more time is required before a difference in mRNA levels is converted into a difference in the abundance of the cognate proteins. Nevertheless, these results raise the possibility that some of the hits may be due to secondary effects, a situation that may explain the larger number of hits obtained for hnRNP C and K. A systematic analysis will be required to determine the full extent of cross talks between different RBPs.

Despite a slight bias in hits for motifs representing binding sites for some hnRNP proteins, additional features beyond the mere presence of binding sites are clearly required for an hnRNP protein to impact alternative splicing. A more extensive examination of the structural environment, sequence, and position of binding motifs relative to splice sites may reveal additional insights into the requirements for splicing control. Moreover, the fact that a majority of splicing units is controlled in a cell line-specific manner indicates that binding sites for a specific hnRNP protein are not sufficient to impose regulation in all cellular contexts. Hit differences between cell lines may reflect differences in the abundance or nuclear availability of given hnRNP proteins or the dominance of other factors implicated in the combinatorial network regulating specific ASEs.

DISCUSSION

Our study is the first to examine the impact of downregulating several mammalian RBPs in multiple cell lines (Fig. (Fig.1).1). This study was conducted with a unique RT-PCR screening technology that provides high sensitivity in the identification of the validated targets, thereby offering a robust experimental system for elucidating the mechanism of hnRNP function in alternative splicing.

Because hnRNP proteins bind RNA, it is likely that the effects on splice site selection result from a direct interaction with the target pre-mRNAs. However, it is possible that a change in the relative abundance of splice isoforms may be achieved by differential mRNA stability, and many hnRNP proteins shuttle to the cytoplasm where they might affect mRNA turnover. In addition, because many RBPs are themselves alternatively spliced, changing the expression of an hnRNP protein may affect the alternative splicing and hence the activity of other RBPs in splicing control. Nevertheless, by examining the impact of reducing the abundance of individual hnRNP proteins we have provided a first glimpse of the complex contribution of hnRNP proteins to the production of splice isoforms in apoptotic genes, and our work sets the stage for a more detailed characterization of the mechanisms of action of the individual hnRNP proteins that target and control alternative splicing and for a more extensive characterization of the network of splicing factors that impact apoptosis.

Downregulating the expression of individual hnRNP proteins affected alternative splicing in a very specific manner, with little overlap in the identity of the targeted events. An hnRNP protein that controls a specific ASE in one cell line does not necessarily affect the same splicing event in other cell lines. Although we have identified 10 splicing events that are controlled by the same hnRNP proteins in all three cell lines, many ASEs are regulated by hnRNP proteins in a cell line-specific manner. Therefore, comparable expression and knockdown efficiencies cannot predict whether a specific splicing event will be regulated by the same hnRNP proteins in different cell lines. This may be because the activity of an hnRNP protein is modulated posttranslationally to affect its cellular distribution and activity (23, 58, 61). Alternatively, other proteins may antagonize its binding and/or activity. Given the combinatorial nature of alternative splicing control, changes in the relative contribution of different RBPs may change considerably in different cellular environments. This variation may offer more flexibility in the way alternative splicing can be regulated, ultimately increasing the composition of splice isoforms from different genes to expand phenotypic diversity.

Supplementary Material

[Supplemental material]

Acknowledgments

We thank Johanne Toutant for preliminary RNA interference assays and Western analysis. We thank Marco Blanchette for helpful comments on the manuscript. We thank Michiyuki Yamada, Doug Black, Karol Bomsztyk, and Mike Mueckler for antibodies.

This study was supported through funding from Genome Canada, Genome Quebec, and the Canadian Institutes in Health Research (B.C.). S.A.E. is a Chercheur-Boursier Senior of the FRSQ. B.C. is the Canada Research Chair in Functional Genomics.

Footnotes

[down-pointing small open triangle]Published ahead of print on 21 July 2008.

Supplemental material for this article may be found at http://mcb.asm.org/.

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