hnRNP L Is a CaRRE1 Binding Factor
To identify the protein factor(s) binding to the CaRRE1 element of the STREX exon, we carried out a UV cross-linking assay with the upstream 3′-splice site of the middle exon of the splicing reporter DUP175ST (), which harbors the CaRRE1 (16
). In HeLa nuclear extracts the wild type RNA transcript was cross-linked mainly to three protein bands of about 55, 65, and 110 kDa, respectively (, right
, lane 1
). Mutation of the CaRRE1 element mainly reduced the 65- and 110-kDa bands (lane 2
). Similarly cross-linked bands were observed in HEK293T nuclear extracts (lanes 3
Immunoprecipitation of the cross-linked proteins in the HEK293T nuclear extracts with antibodies against hnRNP L (lane 5), hnRNP LL (lane 6), or PTB (lane 7) indicates that the 65-kDa band contains hnRNP L, and the 55-kDa band contains PTB. hnRNP LL is not observed in the precipitates even with twice the nuclear extract input (lane 6).
To examine the CaRRE1-binding proteins in depolarized cells, we carried out a similar UV cross-linking assay with nuclear extracts prepared from non-treated or KCl (50 mM)-depolarized GH3
cells (). The pattern of the cross-linked major bands was similar to that in HeLa and HEK293T nuclear extracts. Interestingly, the ratio of the 65- and 55-kDa bands appears increased by about 10% upon depolarization (compare lanes 1
and the graph
on the right
). The cross-linked 65-kDa products was immunoprecipitated by anti-hnRNP L as well (lanes 3
) but not by anti-hnRNP F/H (lane 5
) or anti-hnRNP LL (lanes 6
). Taken together with the previous finding that hnRNP L selectively binds CA repeat elements and controls alternative splicing (26
), these data suggest that hnRNP L is a CaRRE1 binding factor.
An hnRNP L High Affinity CA Repeat Element Is Sufficient to Mediate CaMKIV- and hnRNP L Repression of Splicing in a Location-dependent Way
To isolate the role of hnRNP L and CA repeats from the influence of non-CA nucleotides of CaRRE1, we tested the effect of known hnRNP L binding simple CA repeats in CaMKIV-regulated splicing. We first replaced 17 nt of the CaRRE1 with a high or low affinity sequence of hnRNP L (27
) to make splicing reporters L1 and L1m (). These reporter plasmids were each cotransfected with constitutively active CaMKIV-dCT (IV) or kinase-dead mutant (CaMKIV-dCTK75E, IVm) into HEK293T cells, and inclusion of the middle exon was measured by semiquantitative RT-PCR (PCR for 25 cycles). Exon inclusion levels of both L1 and L1m reporters were similar to the DUP175ST when co-expressed with CaMKIVm (, lanes 1
, and 5
), suggesting that the 17-nt L1 and L1m elements here are not constitutive splicing silencers. However, when co-expressed with CaMKIV, exon inclusion levels of the L1 reporter were reduced by 18% (, compare lanes 3
), similarly as the CaRRE1 reporter DUP175ST (lanes 1
and 2). In contrast, the L1m reporter did not respond to CaMKIV (lanes 5
). Thus, the hnRNP L high affinity CA repeat element can specifically replace CaRRE1 in response to CaMKIV.
Effect of CaRRE1 replacement by an hnRNP L-high affinity CA repeat element in CaMKIV-regulated splicing
To determine whether the L1 element is sufficient to mediate CaMKIV repression of exon inclusion outside of the 3′-splice site, we placed 20 nt of the original L1 sequence inside the middle exon or in the flanking introns of the CaMKIV-nonresponsive DUP175 (16
) and assayed its response to CaMKIV co-expression (, reporters L2–L5
). The middle exon was strongly repressed by CaMKIV only in the L3 reporter that contains the CA repeat in the exon nearby the upstream 3′-splice site (27% reduction, , lanes 5
) but not in any of the other reporters L2, L4, and L5 that contain CA repeats at other locations. Moreover, the repression was abolished by A to G mutations in the CA repeats (L3m
, lane 8
). In comparison to CaMKIV, coexpressed protein kinase A barely reduced the exon inclusion of the L3 reporter (lane 14
). Therefore, the high affinity hnRNP L binding CA repeat element confers CaMKIV-specific repression of exon inclusion in a location-dependent way.
The hnRNP L high affinity CA repeat element in CaMKIV- and hnRNP L-regulated splicing and its location dependence
To determine whether hnRNP L is also able to repress exon inclusion through the CA repeats, we overexpressed hnRNP L-FLAG (L
) with the vector DUP175 or the CA repeat-containing splicing reporters L1-L5 () with YB-1 (Y
), another splicing factor known to interact with A/C-rich elements (49
), as a control. Similar to CaMKIV, hnRNP L-FLAG coexpression strongly repressed inclusion of the middle exon of reporters L1 and L3 (lanes 4
) but not of DUP175, L2, L4, and L5 (lanes 2
, and 16
), neither of the CA repeat mutants (lanes 6
). In contrast, YB-1 coexpression did not change the splicing of any reporter (Y
, odd-numbered lanes
). Therefore, overexpressed hnRNP L specifically represses exon inclusion through the CA repeats, with the same location dependence as CaMKIV.
hnRNP L Mediates CaMKIV Repression of Splicing through the hnRNP L High Affinity CA Repeat Element Nearby the 3′-Splice Site
To determine whether hnRNP L is essential for the CaMKIV-regulated splicing, we examined the response of reporter L3 to CaMKIV without or with hnRNP L depletion by RNA interference (RNAi, ). Exon inclusion of this L3 reporter was again reduced by CaMKIV in cells without hnRNP L depletion (, lanes 1–4). In contrast, when the endogenous hnRNP L was effectively depleted by RNAi (11% protein left, lane 5, compared with controls), the L3 reporter lost its response to CaMKIV (96% ± 1.0 exon inclusion, n = 4, lane 5). Importantly, coexpression of the exogenous hnRNP L-FLAG protein in the RNAi cells restored the CaMKIV repression of the middle exon inclusion (lane 6). Thus, the loss of CaMKIV repression of exon inclusion by RNAi is indeed due to the specific depletion of hnRNP L.
Role of hnRNP L in CaMKIV-regulated splicing through the hnRNP L high affinity CA repeats
To confirm hnRNP L binding to the CA repeats as to the CaRRE1, we cross-linked RNA transcripts containing the CA repeats or its mutant with HeLa or HEK nuclear extracts. The wild type transcript was strongly cross-linked to an ~65-kDa protein (, lanes 2 and 6). Mutating A to G in the CA repeats almost abolished this protein band (lanes 3 and 7). Importantly, this protein was specifically immunoprecipitated by the antibody against hnRNP L (L, lanes 4 and 8) but barely by the control anti-U2AF65 antibody (C, lanes 5 and 9). Thus, hnRNP L interacts with the RNA probe in a CA repeat-dependent way. Taken together, the data from the RNAi/rescue and UV-cross-linking experiments () strongly support hnRNP L as an essential factor mediating CaMKIV-regulated splicing through the hnRNP L highly affinity CA repeat element nearby the 3′-splice site.
hnRNP L Also Mediates CaMKIV-regulated Splicing through Short CA Repeats from Alternative Exons Nearby the 3′-Splice Site
To determine whether shorter CA repeats from alternative exons other than the artificially selected hnRNP L high affinity sequence are also sufficient to mediate CaMKIV repression of exon inclusion, we examined several exonic (CA)3–4
elements of human alternative exons from the ASAPII data base (50
). Four elements with their flanking A/C-rich sequences were transferred to the same location of the DUP175 as in their endogenous exons and assayed for their responses to CaMKIV (). Three of four tested sequences caused CaMKIV reduction of exon inclusion, as examplified in gels for the GALNT11 and PTEN2 elements (, lanes 1
and lanes 5
). Importantly, mutations of the nucleotide A to G in the CA repeats abolished the responses to CaMKIV (lanes 3
7 and 8
). Therefore, shorter CA repeat elements with adjacent A/C-rich sequences nearby the 3′-splice site of alternative exons are also sufficient to mediate CaMKIV-regulated splicing.
Role of hnRNP L in CaMKIV-regulated splicing through short CA repeats from alternative exons
Similar to that for the hnRNP L high affinity CA repeat element (), UV cross-linking and immunoprecipitation experiments indicate that hnRNP L binds the reporter RNA probes, and the CA repeats are critical (). Moreover, similar tests with the PTEN2 reporter in RNAi/rescue experiments also support hnRNP L as an essential factor in the CaMKIV-regulated splicing (). Therefore, hnRNP L also mediates CaMKIV-regulated splicing through the shorter CA repeats from endogenous alternative exons nearby the 3′-splice site. Taken together, the tests with hnRNP L high affinity CA repeats and the shorter ones from alternative exons strongly support that, at or in close proximity to the 3′-splice site, the simple CA repeats and hnRNP L are sufficient to confer CaMKIV response on an otherwise non-responsive heterologous gene.
RNA Interference of hnRNP L Expression Alters Endogenous Exon Inclusion and Response to Membrane Depolarization or CaMKIV
To determine whether hnRNP L is essential for depolarization regulation of endogenous exons, we examined endogenous splice variants in GH3
cells where we efficiently knocked down the hnRNP L protein with the specific shRNA as used in and (, upper panel
). The rat alternative exons of the GALNT11 and PTEN2 genes do not contain the same CA repeats as their human homologues, and their alternative splicing was not detectable in this rat cell line; however, we were able to analyze the STREX exon of the Slo
gene as in the previous report (16
). STREX inclusion was reduced from 33 to 18% by depolarization in cells without hnRNP L depletion (, middle panel
, lanes 1
), similarly as reported (16
). In cells depleted of hnRNP L (lane 3
), STREX inclusion was increased to 47%, indicating that hnRNP L is a repressor of STREX exon inclusion, consistent with its cross-linking to the CaRRE1 (). In these hnRNP L-depleted cells, depolarization still repressed the STREX exon inclusion (compare lanes 3
). However, the relative reduction of exon inclusion by depolarization in these hnRNP L-depleted cells (32%, with 15% of net reduction divided by 47%, lanes 3
) is significantly less than that without hnRNP L depletion (45%, with 15% of net reduction divided by 33%, lanes 1
). We also analyzed the human endogenous exons of the GALNT11 and PTEN2 genes in HEK293T cells and found that the GALNT11 exon is alternatively spliced in this cell line and regulated by hnRNP L and CaMKIV ().
Role of hnRNP L in depolarization- or CaMKIV-regulated endogenous exons
The GALNT11 exon is included in about 25% of transcripts in HEK293T cells when CaMKIVm is expressed (, lane 1). This level was reduced more than half by CaMKIV (lane 2). Interestingly, the reduction was abolished upon knockdown of hnRNP L with the shL plasmid (lane 3). Importantly, the CaMKIV effect was rescued by coexpressed hnRNP L-FLAG (lane 4). Therefore, hnRNP L indeed mediates CaMKIV regulation of the endogenous GALNT11 exon in HEK293T cells.
CaMKIV Enhances hnRNP L Phosphorylation and Its Binding to CaRRE1
To determine how hnRNP L is regulated by CaMKIV, we examined both the protein expression levels and phosphorylation of hnRNP L. No significant changes in the hnRNP L total protein level by depolarization or CaMKIV were observed in GH3 cells or HEK293T cells, respectively. However, a consistent change in hnRNP L binding to CaRRE1 and its phosphorylation with CaMKIV expression was observed ().
hnRNP L is a phosphoprotein with its CaRER1 binding activity regulated by CaMKIV and sensitive to treatment with phosphatases
We first examined the binding of endogenous hnRNP L to CaRRE1 with expressed CaMKIVm or CaMKIV in UV cross-linking assays (). Relative to the cross-linked PTB and the corresponding protein levels in the same gels, the hnRNP L binding to CaRRE1 in CaMKIV-expressed cells was increased about 30% without an apparent change in total protein level.
We next in vivo labeled hnRNP L-FLAG with [32P]-orthophosphoric acid in HEK293T cells and immunoprecipitated it to examine its phosphorylation levels in response to CaMKIV expression (). The total hnRNP L-FLAG protein signal in the Western blot was increased about four times by CaMKIV, likely due to CaMKIV enhancement of this exogenous gene expression through its CMV promoter in the vector; however, the 32P phosphorylation signal was increased about eight times in the same gel. Therefore, relative to its total protein signal, the level of hnRNP L-FLAG phosphorylation was doubled by the expressed CaMKIV.
To determine whether the hnRNP L phosphorylation in CaMKIV-expressed cells is critical for its binding to the CaRRE1, the HEK293T cell nuclear extract containing coexpressed hnRNP L-FLAG was pretreated with λ-protein phosphatase or calf intestinal alkaline phosphatase, and hnRNP L binding to CaRRE1 was examined with UV cross-linking and/or immunoprecipitation (). Relative to the loaded protein levels of the same gels/lanes, hnRNP L-FLAG binding to CaRRE1 was reduced ~30% by either λ-protein phosphatase or calf intestinal alkaline phosphatase treatment. Therefore, the hnRNP L-FLAG binding to CaRRE1 was sensitive to dephosphorylation by phosphatases. Collectively these data support that hnRNP L binding to the CaRRE1 can be regulated by CaMKIV through phosphorylation. Taken together with the above evidence, we conclude that hnRNP L is an essential component in CaMKIV-regulated alternative splicing through CA repeats.