Autoregulatory circuit of human rpL3 expression requires hnRNP H1, NPM and KHSRP

Abstract

Alternative pre-mRNA splicing (AS) is a major mechanism that allows proteomic variability in eukaryotic cells. However, many AS events result in mRNAs containing a premature termination codon, which are degraded by nonsense-mediated mRNA decay (NMD) pathway. We have previously demonstrated that human rpL3 autoregulates its expression through the association of AS with NMD. In fact, overexpression of rpL3 promotes downregulation of canonical splicing and upregulation of alternative splicing that produces an NMD-targeted mRNA isoform. The result of these events is a decreased production of rpL3. We have also identified heterogeneous nuclear ribonucleoprotein (hnRNP) H1 as a splicing factor involved in the regulation of rpL3 alternative splicing and identified its regulatory cis-elements within intron 3 transcript. Here, we report that NPM and KHSRP are two newly identified proteins involved in the regulation of rpL3 gene expression via AS-NMD. We demonstrate that hnRNP H1, KHSRP and NPM can be found associated, and present also in ribonucleoproteins (RNPs) including rpL3 and intron 3 RNA in vivo, and describe protein-protein and RNA-protein interactions. Moreover, our data provide an insight on the crucial role of hnRNP H1 in the regulation of the alternative splicing of the rpL3 gene.

Figure 1.

In vivo binding of NPM, KHSRP, hnRNP H1 and rpL3. NPM or KHSRP were specifically immunoprecipitated from HeLa cells extracts with antibodies against the endogenous NPM and KHSRP. Immunoprecipitates were separated by SDS–PAGE and immunoblotted with antibodies versus the indicated proteins. Note the absence of signal in IgG immunocomplex. Results illustrated in Figures 1–6 are representative of three independently performed experiments.

Figure 2.

Analysis of the interactions between hnRNP H1, NPM, KHSRP and rpL3. Western blotting (WB) of GST pull-down experiments. Fifty microgram of GST-tagged proteins (GST-NPM, GST-rpL3 and GST-hnRNP H1) or GST (control) were immobilized on glutathione-sepharose beads and incubated with 20 μg of His-hnRNP H1, His-NPM or His-KHSRP. The eluted proteins were then analyzed by immunoblot with antibodies anti-hnRNP H1, anti-NPM and anti-KHSRP. Note the absence of signal in control GST pull-down preparations.

Figure 3.

Analysis of the interactions of intron 3 of rpL3 pre-mRNA with NPM and KHSRP, in vivo and in vitro. (A) In vivo binding of intron 3 transcript to NPM and KHSRP. RT–PCR analysis, by using primers against rpL3 intron 3 transcript and rpL7a RNA transcript, of RNA extracted from the NPM, KHSRP, hnRNP H1 (control) and IgG immunocomplexes. Note the absence of signal in IgG immunocomplex. (B) In vitro binding of intron 3 transcript to hnRNP H1, NPM, KHSRP and rpL7a. WB of RNA pull-down experiments, using adipic acid dehydrazide agarose beads coated with intron 3 transcript or unrelated RNA (Ctr RNA) incubated with purified proteins His-hnRNP H1, His-NPM, His-KHSRP and His-rpL7a.

Figure 4.

(A) Effects of KHSRP overexpression on rpL3-a mRNA levels in HeLa cells. Representative RT–PCR analysis of total RNA from CHX-treated HeLa cells untransfected or transfected with increasing amounts of Flag-KHSRP. (B) Effects of KHSRP overexpression on rpL3-a mRNA levels in L3–8 cells. Representative RT–PCR analysis of total RNA from CHX-treated L3–8 cells untransfected or transfected with Flag-KHSRP, non-induced (NI) or induced (I) for HA-rpL3 expression. (C) Effects of RNAi-mediated depletion of KHSRP on rpL3-a mRNA levels in L3–8 cells. Representative RT-PCR analysis of total RNA from CHX-treated L3–8 cells untransfected or transfected with KHSRP-siRNA, NI or induced I for HA-rpL3 expression from the same samples. The levels of rpL3-a mRNA were quantified by PhosphorImager (Bio-Rad) and normalized to β-actin levels.

Figure 5.

(A) Effects of RNAi-mediated depletion of hnRNP H1 on rpL3-a mRNA levels in L3–8 cells. Representative RT-PCR analysis of total RNA from CHX-treated L3–8 cells, untransfected or transfected with hnRNP H1-siRNA, NI or induced I for HA-rpL3 expression. (B) Effects of RNAi-mediated depletion of hnRNP H1 and KHSRP overexpression on rpL3-a mRNA levels in L3–8 cells. Representative RT–PCR analysis of total RNA from CHX-treated L3–8 cells untransfected or cotransfected with hnRNP H1-siRNA and Flag-KHSRP, NI or induced I for HA-rpL3 expression. The levels of rpL3-a mRNA were quantified by PhosphorImager (Bio-Rad) and normalized to β-actin levels.

Figure 6.

(A) Effects of NPM overexpression on rpL3-a mRNA levels in HeLa cells. Representative RT–PCR analysis of total RNA from CHX-treated HeLa cells untransfected or transfected with increasing amounts of HA-NPM. (B) Effects of overexpression of NPM on rpL3-a mRNA levels in L3–8 cells. Representative RT–PCR analysis of total RNA from CHX-treated L3–8 cells untransfected or transfected with HA-NPM, NI or I for HA-rpL3 expression. (C) Effects of silencing of NPM on rpL3-a mRNA levels in L3–8 cells. Representative RT–PCR analysis of total RNA from CHX-treated L3–8 cells untransfected or transfected with siRNA against NPM, NI or I for HA-rpL3 expression. The levels of rpL3-a mRNA were quantified by PhosphorImager (Bio-Rad) and normalized to β-actin levels.

Figure 7.

Schematic representation of proposed rpL3 feedback regulation. (A) In response to cell requirement for an efficient production of rpL3, NPM represses the alternative splicing of rpL3 gene. NPM, by interacting with hnRNP H1 and intron 3 pre-mRNA, prevents the binding of hnRNP H1 with G3 and G6 motifs. (B) Upon accumulation, rpL3 interacts with NPM. As consequence of this interaction, NPM is released from the RNP complex. The association of rpL3 with KHSRP and other transacting proteins induces a rearrangement of the interactions within the RNP complex that favor the interaction of hnRNP H1 with enhancer unit G3 + G6 within intron 3 transcript. Finally, the reassembled RNP complex promotes the selection of the 3′-splice site in intron 3 transcript.