Unknown,Transcriptomics,Genomics,Proteomics

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HnRNPLL is a global regulator of alternative splicing in activated T cells

ABSTRACT: HnRNPLL was identified as a critical regulator of CD45 alternative splicing in a lentiviral shRNA screen. RNAi-mediated depletion of hnRNPLL eliminated the activation-induced induced transition from the CD45RA to the CD45RO isoform. HnRNPLL is induced during the process of T cell activation, raising the possibility that it regulates a broad program of alternative splicing in activated T cells. To test this possibility and to identify additional potential targets of hnRNPLL, we performed exon array analysis on RNA isolated from five cellular conditions: 1) activated peripheral CD4+ T cells, 2) peripheral CD4+ T cells infected with a control shRNA directed against GFP, 3) peripheral CD4+ T infected with an shRNA directed against hnRNPLL, 4) naÃ¯ve cord blood CD4+ T cells, and 5) cord blood CD4+ T cells that had been activated with anti-CD3 and anti-CD28 for 24 hours. The RNA was hybridized to Affymetrix human exon arrays and the hybridization signals were analyzed with XRAYTM software (Biotique). Using stringent filters for non-expressed probesets, we identified 132 genes that showed significant alternative exon usage (p<0.01) in response to hnRNPLL knockdown, but not in response to shGFP infection. Of these 132 genes, 36 also showed significant alternative exon usage in response to activation of cord blood cells, which results in an approximate 5-fold increase in hnRNPLL expression. We thus conclude that induction of hnRNPLL represents a mechanism by which cells can rapidly shift their transcriptomes during the process of T cell activation. This SuperSeries is composed of the following subset Series: GSE11832: naive/activated ANOVA group GSE11833: LL-sh4/uninfected/shGFP ANOVA group

ORGANISM(S): Homo sapiens

SUBMITTER: Shalini Oberdoerffer

PROVIDER: E-GEOD-11834 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Regulation of CD45 alternative splicing by heterogeneous ribonucleoprotein, hnRNPLL.

Oberdoerffer Shalini S Moita Luis Ferreira LF Neems Daniel D Freitas Rui P RP Hacohen Nir N Rao Anjana A

Science (New York, N.Y.) 20080710 5889

The transition from naïve to activated T cells is marked by alternative splicing of pre-mRNA encoding the transmembrane phosphatase CD45. Using a short hairpin RNA interference screen, we identified heterogeneous ribonucleoprotein L-like (hnRNPLL) as a critical inducible regulator of CD45 alternative splicing. HnRNPLL was up-regulated in stimulated T cells, bound CD45 transcripts, and was both necessary and sufficient for CD45 alternative splicing. Depletion or overexpression of hnRNPLL in B and ...[more]

PMID: 18669861

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Project description:Though T cell expansion and effector differentiation are triggered and, perhaps, maintained by antigen, the proliferative behaviors of CD4+ and CD8+ T cells responding to timed antigen presentation have rarely been compared side by side. Proliferation and effector differentiation of TCR transgenic and polyclonal T cells were analyzed following transient and continuous TCR signals. We found CD4+ T cell proliferation to be dependent on prolonged antigen presence, whereas CD8+ T cells were able to divide and differentiate into effector cells in the absence of it. We excluded CD4+ T cell proliferation to be abrogated by coinhibitory signals or the lack of inflammatory stimuli and found that autonomous proliferation of CD8* T cells was independent of any MHC class I signals. Gene expression analyses illustrated differences in global gene transcription between the two subsets following stimulation periods of different lengths. These T cell data reflect the MHC class difference in that class II but not class I molecules were stabilized on activated DCs in vivo, suggesting a coevolution of MHC molecules and their respective T cell subsets. Samples 1-12: Analysis on day 2. Purified CD4+ AND-TCR transgenic cells andÂ CD8+ OT1-TCR transgenic cells were separately stimulated with anti-CD3 and anti-CD28 antibodies. 48 hours later, the cells were sorted again to a purity of >99 %. Extracted total RNA was amplified twice and hybridized on Affymetrix Mouse 430A2 microarrays. First, we analysed the changes of the CD4+ and CD8+ T cells after stimulation. Second, we compared the differences of the changes between the two cell types after stimulation. For each of the four groups (CD4+ and CD8+, stimulated and unstimulated), we analysed three independent biological replicates. Samples 13-28: Analysis on day 5. AND and OT1 TCR-transgenic T cells were prepared as described before, but transferred into mice that do not or do present their respective antigens. 72 hours later, the cells were FACS-sorted twice to >99 % purity, directly into Trizol. For each of the six groups (CD4+ and CD8+, unstimulated, transient (2 days) and continuous (5 days) stimulation), three independent biological replicates were analyzed, except for CD4+ unstimulated and CD4+ transient, with two replicates each.

Dataset Information

HnRNPLL is a global regulator of alternative splicing in activated T cells

Publications

Regulation of CD45 alternative splicing by heterogeneous ribonucleoprotein, hnRNPLL.

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