Promotion of exon 6 inclusion in HuD pre-mRNA by Hu protein family members

Abstract

The Hu RNA-binding protein family consists of four members: HuR/A, HuB, HuC and HuD. HuR expression is widespread. The other three neuron-specific Hu proteins play an important role in neuronal differentiation through modulating multiple processes of RNA metabolism. In the splicing events examined previously, Hu proteins promote skipping of the alternative exons. Here, we report the first example where Hu proteins promote inclusion of an alternative exon, exon 6 of the HuD pre-mRNA. Sequence alignment analysis indicates the presence of several conserved AU-rich sequences both upstream and downstream to this alternatively spliced exon. We generated a human HuD exon 6 mini-gene reporter construct that includes these conserved sequences. Hu protein over-expression led to significantly increased exon 6 inclusion from this reporter and endogenous HuD. Studies using truncated and mutant HuD exon 6 reporters demonstrate that two AU-rich sequences located downstream of exon 6 are important. RNAi knockdown of Hu proteins decreased exon 6 inclusion. An in vitro splicing assay indicates that Hu proteins promote HuD exon 6 inclusion directly at the level of splicing. Our studies demonstrate that Hu proteins can function as splicing enhancers and expand the functional role of Hu proteins as splicing regulators.

Figure 1.

Hu splice variants (sv) and alternative splicing of HuD in rat brain and CA77 cells. (A) Schematic diagram of the Hu exon-intron structure. The RRM1 domain is located in exon 2 and part of exon 3. The RRM2 domain is located in part of exon 3, exon 4 and part of exon 5. The hinge domain is located in part of exon 5, exon 6 and part of exon 7. RRM3 is located on exon 7. (B) Splice variants of Hu proteins. Schematic diagram showing different splice variants of Hu protein family members. (C) RT-PCR analysis of HuD alternative splicing using RNA isolated from rat brain and CA77 cells. The HuD splice variants are indicated on the right.

Figure 2.

Genomic sequences surrounding exon 6 of HuD are conserved across species. The HuD genomic sequence from Homo sapiens (AL592182), Mus musculus (AL627425) and Rattus norvegicus (NW_047718) were aligned and compared. Conserved sequences are shaded in black. The bold lines under the sequence represent the conserved AU-rich sequences. The nucleotides of exon 6 are indicated in capital letters.

Figure 3.

Hu proteins promote HuD exon 6 inclusion. (A) A schematic diagram of the HuD splicing reporter construct E6. The two potential splicing products are indicated. (B) E6 reporter was co-transfected into HeLa cells with pCDNA3.1 vector, TIAR, or different Hu protein isoforms. Splicing of the reporter was analyzed by RT-PCR on total RNA isolated from the transfected cells (top). The percentage of exon 6 inclusion is indicated below the RT-PCR gel. Expression of co-transfected proteins was analyzed by western blot assay (bottom). γ-Tubulin was used as a loading control. The molecular markers for DNA or protein size are indicated on the left of the gels. (C) Graphic representation of RT-PCR results for exon 6 inclusion of the E6 reporter shown in (B), with error bars indicating standard deviations. (D) F9 cells were transfected with pCDNA3.1 vector or HuC protein. Splicing of the endogenous HuD was analyzed by RT-PCR on total RNA isolated from the transfected cells (top). The percentage of HuD sv4 production is indicated below the RT-PCR gel. Expression of the over-expressed protein was analyzed by western blot (bottom). γ-tubulin was used as a loading control.

Figure 4.

Minimal sequences required for Hu-mediated inclusion of exon 6. (A) Schematic diagrams depicting the six truncated E6 reporters that contain varying lengths of sequence downstream of exon 6. (B) The pcDNA3.1 or HuC sv2 expression vector was co-transfected with the truncated reporters into HeLa cells. Splicing was analyzed using RT-PCR shown in the top panel. Expression of HuC sv2 was analyzed by western blot assay shown in the bottom panel. (C) Graphic representation of the RT-PCR results shown in (B). (D) Schematic diagram of the truncated E6 reporter E6-T7. Splicing of this reporter in HeLa cells in the absence or presence of HuC sv2 over-expression was analyzed using RT-PCR shown in the right panel. Expression of HuC sv2 was analyzed by western blot assay shown in the bottom panel.

Figure 5.

Identification of two AU-rich elements important for Hu-mediated exon 6 inclusion. (A) Schematic diagram of the wild type (WT) and mutant E6-T5 reporters. The sequences in the AT-rich region in the wild type and mutant reporters are shown. (B) Wild type or mutant reporters were co-transfected with pcDNA3.1 or HuC sv2 expression vector. Splicing was analyzed by RT-PCR. Expression of HuC sv2 shown in the western blot analysis. (C) Graphic representation of the RT-PCR results shown in (B).

Figure 6.

Decreased inclusion of HuD exon 6 in CA77 cells with reduced level of Hu proteins. E6-T5 reporter was co-transfected with shRNA plasmid of eGFP, HuB-1, HuB-2, HuC-1, HuC-2 or HuD into CA77 cells. (A) Splicing of the reporter was analyzed by RT-PCR. (B) RT-PCR analysis of endogenous HuB, HuC and HuD expression in CA77 cells transfected with shRNA plasmids. The β-actin in the shRNA-treated cells used as a control. (C) To test the knockdown efficiency of the shRNA plasmids, they were co-transfected with Hu expression plasmid into CA77 cells. Expression of the over-expressed Hu with eGFP or Hu shRNA plasmids are shown in the western blot analysis.

Figure 7.

Hu proteins bind to the AU-rich elements downstream of HuD exon 6. (A) The sequences of the wild type and mutant RNA oligonucleotides are shown. (B) RNA gel mobility shift analysis. The wild type (lanes 1–7) or mutant (lanes 8–14) RNA was incubated with no protein (lanes 1 and 8), increasing amounts (2, 10 and 50 ng) of GST protein alone (lanes 2–4 and 9–11) or GST-HuC sv4 (lanes 5–7 and 12–14).

Figure 8.

HuC promotes the inclusion of HuD exon 6 in an in vitro splicing assay. (A) A schematic diagram of the construct used to generate the RNA splicing substrate for the in vitro splicing assay. The black arrows below the diagram indicate the position of primers used for RT-PCR analysis. (B) Lanes 1–5 indicate the splicing activity of the wild-type transcript in the presence of buffer alone, increasing amounts (1 and 2 µg) of GST alone, or increasing amounts GST-HuC sv4. Lanes 6–10 indicate the splicing activity of the Mut 1 + 2 transcript under the same conditions. (C) Graphic representation of the RT-PCR results shown in (B).