Research ArticleFibrosis

Nuclear hyaluronidase 2 drives alternative splicing of CD44 pre-mRNA to determine profibrotic or antifibrotic cell phenotype

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Science Signaling  21 Nov 2017:
Vol. 10, Issue 506, eaao1822
DOI: 10.1126/scisignal.aao1822
  • Fig. 1 Schematic of CD44 pre-mRNA and alternatively spliced CD44s and CD44v7/8 mRNAs.

    (A) Intron-exon structure of CD44 pre-mRNA. The 5′ common region exons 1 to 5 and 3′ common region exons 15 to 17 and 19 are present in all CD44 splice variants. Exon 18 is removed before translation, and exons 6 to 14 are differentially expressed in different CD44 variant isoforms. (B) CD44s mRNA contains only the common exons 1 to 5, 15 to 17, and 19. All variably expressed exons are removed from this isoform by splicing. (C) The CD44v7/8 mRNA contains the same common region exons as CD44s plus the addition of the variably expressed exons 11 and 12. CD44v7 mRNA contains the same common region exons as CD44s plus the variably expressed exon 11. CD44v8 mRNA contains the same common region exons as CD44s plus the variably expressed exon 12. Exons 11 and 12 encode an extracellular stem region.

  • Fig. 2 CD44s expression is dependent on the splicing regulators SRSF2 and SRSF5.

    Fluorescence microscopy showing enhanced green fluorescent protein (EGFP) or red fluorescent protein (dsRED) expression in proximal tubular epithelial cell (PTEC) (HK-2) cell lines stably expressing (A) pRG6-CD44v7 or (B) pRG6-CD44v8 after transfection with a scrambled small interfering RNA (siRNA) or scrambled siRNAs directed against SRSF2 or SRSF5 in the presence or absence of bone morphogenetic protein 7 (BMP7). Scale bar, 100 μm. (C to H) Expression of SRSF2, CD44s, and CD44v7/8 in untransfected PTECs and in PTEC cell lines stably expressing pRG6-CD44v7 or pRG6-CD44v8 and cotransfected with constructs encoding scrambled siRNAs (white bars) or siRNAs directed against SRSF2 or SRSF5 (black bars). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) showing abundance of (C) SRSF2, (D) CD44s, and (E) CD44v7/8 mRNAs in cells transfected with siRNA targeting SRSF2 (siSRSF2). qRT-PCR showing abundance of (F) SRSF5, (G) CD44s, and (H) CD44v7/8 mRNAs in cells transfected with siRNA targeting SRSF5 (siSRSF5). All data are means ± SE of three independent experiments (n = 3). Statistical analysis is shown as *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001, as determined by Tukey’s posttest. NS, nonsignificant.

  • Fig. 3 BMP7 promotes HYAL2 nuclear translocation and binding to CD44 pre-mRNA.

    (A) Confocal microscopy images showing hyaluronidase 2 (HYAL2) accumulation in growth-arrested primary human lung fibroblasts after BMP7 stimulation. Scale bar, 5 μm. (B) Western blots showing HYAL2 protein abundance in cytoplasmic and nuclear fractions with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and histone H3 as loading controls. The cells were transfected with a construct encoding a siRNA targeting HYAL2 (siHYAL2+) or scrambled control siRNA (siHYAL2), as indicated, before growth arrest and BMP7 treatment. (C) Densitometric analysis of Western blots in (B). (D) RNA immunoprecipitation (RIP) using an antibody directed against HYAL2 followed by qRT-PCR to detect the indicated regions of CD44 transcripts. Intronic regions, CD44iχ; exonic regions, CD44vχ; χ, intron or exon number. Images and blots are representative of three independent experiments using fibroblasts; similar results were obtained using PTEC (HK-2) cells. Data are means ± SE of three independent experiments (n = 3). Statistical analysis is shown as *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001, as determined by Tukey’s posttest.

  • Fig. 4 HYAL2 is a critical regulator of CD44v7/8 alternative splicing.

    Expression of (A) HYAL2, (B) CD44s, and (C) CD44v7/8 by qRT-PCR after BMP7 treatment of primary human lung fibroblasts transfected with scrambled siRNAs (white bars) or siRNAs directed against HYAL2 (siHYAL2, black bars). (D) Western blot showing CD44s (standard CD44) and CD44v7/8 protein abundance after BMP7 treatment of primary human lung fibroblasts expressing siHYAL2. GAPDH was used as a loading control. (E) Densitometric analysis of Western blot in (C). (F to H) qRT-PCR showing expression of (F) HYAL2, (G) CD44s, and (H) CD44v7/8 in PTEC (HK-2) cell lines stably expressing pRG6-CD44v7 or pRG6-CD44v8 and transfected with scrambled siRNAs (white bars) or siRNAs targeting HYAL2 (siHYAL2, black bars). (I and J) Fluorescence microscopy showing EGFP or dsRED in cells expressing (I) pRG6-CD44v7 or (J) pRG6-CD44v8. Scale bars, 100 μm. Images, blots, and graphs are representative of three experiments. Data are means ± SE of three independent experiments (n = 3). Statistical analysis is shown as *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001, as determined by Tukey’s posttest.

  • Fig. 5 HYAL2 displaces SRSF proteins from snRNAs and prevents mature spliceosome formation and splicing initiation.

    (A) Western blot showing SRSF2 (SR splicing factor 2) and SRSF5 after HYAL2 coimmunoprecipitation of nuclear extracts from primary human lung fibroblasts treated with BMP7. (B) qRT-PCR was used for detecting the indicated regions of CD44 transcripts after RIP of SRSF5 from whole-cell extracts of fibroblasts transfected with scrambled siRNAs or siRNAs targeting HYAL2 (siHYAL2) with and without BMP7 treatment. Intronic regions, CD44iχ; exonic regions, CD44vχ; χ, intron or exon number. (C and D) qRT-PCR showing (C) SRSF2 and (D) SRSF5 expression in fibroblasts and PTEC (HK-2) cells transfected with scrambled siRNAs (white bars) or siRNAs targeting HYAL2 (siHYAL2, black bars) with and without BMP7 treatment. (E to G) qRT-PCR for U1, U2, U4, and U6 small nuclear RNAs (snRNAs) after RIP of extracts from BMP7-treated primary human lung fibroblasts using antibodies directed against (E) HYAL2, (F) SRSF2, or (G) SRSF5. Blots represent three independent experiments using fibroblasts; similar results were obtained using PTEC (HK-2) cells. Data are means ± SE of three independent experiments (n = 3). Statistical analysis is shown as *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001, as determined by Tukey’s posttest.

  • Fig. 6 Model for alternative splicing that generates profibrotic and antifibrotic isoforms of CD44.

    (A) Schematic of CD44 pre-mRNA from intron 10 to intron 12, indicating putative 5′ splice sites (5′SS; red triangles) and 3′SS (pink triangles). The high density of 3′SS within intron 12 suggests preferential binding of the spliceosome to this intron under basal conditions, favoring splicing activity at these sites. CD44v7/8 pre-mRNA includes exon 11 (v7) and exon 12 (v8). (B) Mechanism of CD44s pre-mRNA splicing. Under basal conditions, there is preference for splicing that favors CD44s expression. Binding of SRSF5 to introns 10 and 12 recruits early spliceosomes (U1/U2) to the 5′SS of intron 10 and the 3′SS of intron 12. SRSF2 facilitates the activity of early spliceosomes. Subsequent recruitment of the mature spliceosome machinery (U4/U6.U5) results in a double-exon-skipping alternative splicing event, wherein introns 10 and 12 are spliced out simultaneously, and exons 11 and 12 and intron 11 are removed. This generates the CD44s transcript, which lacks both exons 11 and 12. Production of CD44s allows establishment of the myofibroblast-stabilizing hyaluronan (HA) coat and signaling through epidermal growth factor receptor (EGFR) after transforming growth factor–β1 (TGF-β1) stimulation. (C) Mechanism of CD44v7/8 alternative splicing. After BMP7 stimulation, there is preference for splicing to increase CD44v7/8 expression. HYAL2 displaces SRSF5 that is bound to the 3′SSs in intron 12 of the CD44 pre-mRNA. HYAL2 concomitantly displaces SRSF5 from U1/U2, preventing initiation of splicing at intron 12. HYAL2 also attenuates SRSF2 and SRSF5 mRNA expression, leading to decreased abundance of these splice factors and reduced availability to bind to components of the early (U1/U2) spliceosome. The mature (U4/U6.U5) spliceosome cannot be recruited, and the double-exon-skipping alternative splicing event is avoided. Consequently, only intron 10 is spliced out, ordinal pre-mRNA processing continues, and CD44v7/8 expression is protected. Production of CD44v7/8 internalizes HA and prevents the formation of the myofibroblast-stabilizing HA coat. The mechanism(s), by which exons 11 and 12 are differentially spliced to yield the CD44v7 and CD44v8 transcripts, has not been identified.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/506/eaao1822/DC1

    Fig. S1. Schematic and confirmation of function for pRG6-CD44v bichromatic minigene reporters.

    Fig. S2. In silico analysis of intronic sequence surrounding exons 11 and 12 of CD44v7 and CD44v8.

    Fig. S3. Confirmation of SRSF2 and SRSF5 knockdown by Western blot.

    Fig. S4. SRSF2 RIP.

    Table S1. Threshold scores for SR proteins (CD44 introns 10, 11, and 12) derived from in silico analysis.

    Table S2. Threshold scores for SR proteins (CD44 exons 11 and 12) derived from in silico analysis.

    Table S3. Individual protein IDs from nuclear HYAL2 coimmunoprecipitation.

    Table S4. In silico analysis of HYAL2 splice factor binding site thresholds.

    Table S5. Primer sequences used for qRT-PCR and RIP.

    Table S6. qRT-PCR cycle threshold values for CD44s and CD44v7/8.

  • Supplementary Materials for:

    Nuclear hyaluronidase 2 drives alternative splicing of CD44 pre-mRNA to determine profibrotic or antifibrotic cell phenotype

    Adam C. Midgley, Sebastian Oltean, Vincent Hascall, Emma L. Woods, Robert Steadman, Aled O. Phillips, Soma Meran*

    *Corresponding author. Email: merans{at}cf.ac.uk

    This PDF file includes:

    • Fig. S1. Schematic and confirmation of function for pRG6-CD44v bichromatic minigene reporters.
    • Fig. S2. In silico analysis of intronic sequence surrounding exons 11 and 12 of CD44v7 and CD44v8.
    • Fig. S3. Confirmation of SRSF2 and SRSF5 knockdown by Western blot.
    • Fig. S4. SRSF2 RIP.
    • Table S1. Threshold scores for SR proteins (CD44 introns 10, 11, and 12) derived from in silico analysis.
    • Table S2. Threshold scores for SR proteins (CD44 exons 11 and 12) derived from in silico analysis.
    • Table S3. Individual protein IDs from nuclear HYAL2 coimmunoprecipitation.
    • Table S4. In silico analysis of HYAL2 splice factor binding site thresholds.
    • Table S5. Primer sequences used for qRT-PCR and RIP.
    • Table S6. qRT-PCR cycle threshold values for CD44s and CD44v7/8.

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    Citation: A. C. Midgley, S. Oltean, V. Hascall, E. L. Woods, R. Steadman, A. O. Phillips, S. Meran, Nuclear hyaluronidase 2 drives alternative splicing of CD44 pre-mRNA to determine profibrotic or antifibrotic cell phenotype. Sci. Signal. 10, eaao1822 (2017).

    © 2017 American Association for the Advancement of Science

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