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Transcription elongation factor ELL2 directs immunoglobulin secretion in plasma cells by stimulating altered RNA processing

Nature Immunology volume 10, pages 1102–1109 (2009)Cite this article

Abstract

Immunoglobulin secretion is modulated by competition between the use of a weak promoter-proximal poly(A) site and a nonconsensus splice site in the final secretory-specific exon of the heavy chain pre-mRNA. The RNA polymerase II transcription elongation factor ELL2, which is induced in plasma cells, enhanced both polyadenylation and exon skipping with the gene encoding the immunoglobulin heavy-chain complex (Igh) and reporter constructs. Lowering ELL2 expression by transfection of heterogenous ribonucleoprotein F (hnRNP F) or small interfering RNA resulted in lower abundance of secretory-specific forms of immunoglobulin heavy-chain mRNA. ELL2 and the polyadenylation factor CstF-64 tracked together with RNA polymerase II across the Igh μ- and γ-gene segments; the association of both factors was blocked by ELL2-specific small interfering RNA. Thus, loading of ELL2 and CstF-64 on RNA polymerase II was linked, caused enhanced use of the proximal poly(A) site and was necessary for processing of immunoglobulin heavy-chain mRNA.

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Figure 1: Immunoglobulin γ2b reporter gene.
Figure 2: Cells stimulated to secrete have more ELL2 mRNA and protein.
Figure 3: ELL2 and CstF-64 'track' together |with RNAP-II across Igh.
Figure 4: ELL2-specific shRNA to interferes with ChIP of CstF-64 at the Igh promoter region.
Figure 5: ELL2 enhances the production of secretory mRNA.
Figure 6: ELL2 influences poly(A) site 'choice'.
Figure 7: ELL2 enhances exon skipping.

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Acknowledgements

We thank A. Shilatifard (Stowers Institute for Medical Research) for monoclonal antibody to ELL2; A. Kornblihtt (University of Buenos Aires) for splicing reporter plasmids; C. Webb (University of Oklahoma) for Igh promoters; J. Fink for assistance in constructing COOH ELL2; S. Eghtesad for cloning ELL2-specific shRNA; J. Heakins for mouse spleen cell chromatin; and B. Junker and Y. Lin for help with statistical analyses. Supported by the US National Institutes of Health (CA86433 to C.M. and AI079047 to L.B.) and the National Science Foundation (MCB-8842725 to C.M.).

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Authors and Affiliations

  1. Department of Immunology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania

    Kathleen Martincic, Serkan A Alkan, Alys Cheatle, Lisa Borghesi & Christine Milcarek

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  1. Kathleen Martincic
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Contributions

K.M. cloned ELL2 segments for Figure 1b and did immunoblot analyses for Figure 2, transfection for Figures 5a, 6 and 7 and in vitro studies for Supplementary Figures 1 and 2 and prepared all figures; S.A.A. did the microarray study; A.C. did the quantitative RT-PCR for Table 1; L.B. provided the splenic B cells for Figures 2c, 3a and 5b; C.M. did the RNA hybridization blot for Figure 2a, the ChIP experiments for Figures 3 and 4 and the quantitative RT-PCR for Figure 5b, and conceived the studies, provided critical guidance and wrote the manuscript with assistance from K.M. and L.B; and all authors reviewed and approved the manuscript.

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Correspondence to Christine Milcarek.

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Supplementary Figures 1–4, Supplementary Tables 1–2 and Supplementary Methods (PDF 509 kb)

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Martincic, K., Alkan, S., Cheatle, A. et al. Transcription elongation factor ELL2 directs immunoglobulin secretion in plasma cells by stimulating altered RNA processing. Nat Immunol 10, 1102–1109 (2009). https://doi.org/10.1038/ni.1786

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