Alternative Splicing Controlled by Heterogeneous Nuclear Ribonucleoprotein L Regulates Development, Proliferation, and Migration of Thymic Pre-T Cells This information is current as of October 2, 2021. Marie-Claude Gaudreau, Florian Heyd, Rachel Bastien, Brian Wilhelm and Tarik Möröy J Immunol published online 20 April 2012 http://www.jimmunol.org/content/early/2012/04/20/jimmun ol.1103142 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2012/04/20/jimmunol.110314 Material 2.DC1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published April 20, 2012, doi:10.4049/jimmunol.1103142 The Journal of Immunology Alternative Splicing Controlled by Heterogeneous Nuclear Ribonucleoprotein L Regulates Development, Proliferation, and Migration of Thymic Pre-T Cells Marie-Claude Gaudreau,*,† Florian Heyd,‡ Rachel Bastien,* Brian Wilhelm,x and Tarik Mo¨ro¨y*,† The regulation of posttranscriptional modifications of pre-mRNA by alternative splicing is important for cellular function, devel- opment, and immunity. The receptor tyrosine phosphatase CD45, which is expressed on all hematopoietic cells, is known for its role in the development and activation of T cells. CD45 is known to be alternatively spliced, a process that is partially regulated by heterogeneous nuclear ribonucleoprotein (hnRNP) L. To investigate the role of hnRNP L further, we have generated conditional hnRNP L knockout mice and found that LckCre-mediated deletion of hnRNP L results in a decreased thymic cellularity caused by a partial block at the transition stage between double-negative 4 and double-positive cells. In addition, hnRNP L2/2 thymocytes Downloaded from express aberrant levels of the CD45RA splice isoforms and show high levels of phosphorylated Lck at the activator tyrosine Y394, but lack phosphorylation of the inhibitory tyrosine Y505. This indicated an increased basal Lck activity and correlated with higher proliferation rates of double-negative 4 cells in hnRNP L2/2 mice. Deletion of hnRNP L also blocked the migration and egress of single-positive thymocytes to peripheral lymphoid organs in response to sphingosine-1-phosphate and the chemokines CCL21 and CXCL12 very likely as a result of aberrant splicing of genes encoding GTPase regulators and proteins affecting cytoskeletal organization. Our results indicate that hnRNP L regulates T cell differentiation and migration by regulating pre-TCR http://www.jimmunol.org/ and chemokine receptor signaling. The Journal of Immunology, 2012, 188: 000–000. hymocytes must pass through a tightly regulated devel- by upregulating both CD4 and CD8 markers to become double- opmental process to mature into effector T cells. First, positive (DP) cells (4–9). These cells then undergo a positive and T early lymphocyte precursors migrate from bone marrow to negative selection to eliminate autoreactive T cells and to produce the peripheral blood and enter the thymus, where they undergo the final single-positive (SP) CD4- and CD8-expressing T effector differentiation (1, 2). The most immature thymocytes, termed cell populations. During these two selection processes, thymo- double negative (DN), are characterized by the absence of both cytes receive different signals from the pre-TCR, the TCR, the by guest on October 2, 2021 CD4 and CD8 coreceptor surface markers and are subdivided coreceptors, and probably other receptors that promote cell sur- based on the expression of CD44 and CD25 into four fractions vival and proliferation (4, 10–16). called DN1 to DN4 (3). In DN3 cells, b-selection takes place, One of the critical factors that regulate the strength of TCR or which assures the selection of cells with a productive rearrange- pre-TCR signaling is the transmembrane tyrosine phosphatase ment of the TCRb gene and the correct assembly of a surface pre- CD45, which is not only expressed on T cells, but is found on TCR complex. From this, DN4 cells emerge and develop further a wide variety of hematopoietic cells, except platelets and eryth- rocytes (17, 18). This protein exerts its regulatory function by modulating the activity of the src kinases Lck and Fyn (18–22). *Institut de Recherches Cliniques de Montre´al, Montre´al, Que´bec H2W 1R7, Can- Multiple isoforms of CD45 can be generated by alternative † ada; De´partement de Microbiologie et Immunologie, Universite´ de Montre´al, Mon- splicing of the variable exons 4–6, also called A, B, and C, which tre´al, Que´bec H3T 1J4, Canada; ‡Institut fu¨r Molekularbiologie und Tumorforschung, x Philipps Universita¨t Marburg, D-35032 Marburg, Germany; and Institut de code for different extracellular domains of the protein (23). The Recherches en Immunologie et Cancer, Universite´ de Montre´al, Montre´al, Que´bec expression of a specific isoform of CD45 is cell-type specific and H3C 3J7, Canada changes during thymocyte development. Immature DPs predom- Received for publication November 2, 2011. Accepted for publication March 22, inantly express CD45RO, which lacks the domains encoded by 2012. exons 4–6, whereas CD4SP or CD8SP cells express the high m.w. This work was supported by a Canadian Institutes of Health Research (CIHR) Ph.D. fellowship (to M.-C.G.), the Institut de Recherches Cliniques de Montre´al, the Ca- isoform CD45RB, which contains the domain encoded by exon 5 nadian Foundation for Innovation, CIHR Operating Grant MOP-86516, and a Canada (24–26). The smaller isoforms of CD45 form predominantly Research Chair (Tier 1; to T.M.). homodimers, whereas the high m.w. isoforms lose this potential, The sequences presented in this article have been submitted to Gene Expression resulting in a less efficient signal transduction (27). A number of Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE33306. studies using CD45-deficient mice have demonstrated a crucial Address correspondence and reprint requests to Dr. Tarik Mo¨ro¨y, Institut de role for this protein, because its absence results in a severely Recherches Cliniques de Montre´al, 110 des Pins West Avenue, Montre´al, Que´bec H2W 1R7, Canada. E-mail address: [email protected] impaired TCR signal transduction and in a differentiation block The online version of this article contains supplemental material. during positive and negative selection that occurs during the dif- Abbreviations used in this article: CT, cycle threshold; DN, double negative; DP, ferentiation of DP thymocytes to mature SP cells (28–30). double positive; GO, gene ontology; hnRNP, heterogeneous nuclear ribonucleopro- Several lines of evidence provided by in vitro studies on al- tein; SP, single positive; S1P, sphingosine-1-phosphate; S1P1, S1P receptor 1; wt, ternative splicing of CD45 reveal the implication of heterogeneous wild-type. nuclear ribonucleoprotein (hnRNP) L in mediating this process. Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 The hnRNP proteins belong to a family of RNA-binding factors www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103142 2 hnRNP L IN T CELL DEVELOPMENT AND MIGRATION that regulate alternative splicing by binding exonic splicing silencer and immediately incubated at 37˚C for the indicated time. Cells were elements, resulting in exon exclusion from the mature mRNA (31). processed for surface and intracellular staining according to standard In the case of hnRNP L, its binding to the exonic splicing silencer procedure. elements 1 sequence in the alternatively spliced CD45 exons Proliferation assays results in their exclusion from the mature, spliced RNA (23, 31). For in vivo proliferation assays, mice were injected i.p. with 200 ml 10 mg/ We show in this study that deletion of hnRNP L in the mouse ml solution 5-bromo-2-deoxyuridine (Sigma-Aldrich) 16 h before sacri- results in a very early block of embryonic development, empha- fice. Thymocytes were stained with specific fluorescent Abs, fixed, and sizing its crucial role in morphogenesis. To study a role of hnRNP L treated with Perm/Wash (BD Biosciences). DNase I (Sigma-Aldrich) in T cell development and function, we have restricted hnRNP L treatment at 30 mg/ml was applied for 1 h at 37˚C. Anti-BrdU FITC conjugated (BD Biosciences) was added for 30 min at room temperature, deletion to the T cell compartment by using a T cell-specific Cre and events were acquired on a LSR (BD Biosciences). recombinase transgene (LckCre). This strategy allowed us to re- veal new and important roles of alternative splicing mediated by RT-PCR hnRNP L in T cell maturation and migration. Total RNA was extracted from thymocytes using RNeasy mini kit (Qiagen), according to the manufacturer’s instructions. For RT-PCR analysis, RNA Materials and Methods was used to prepare cDNA using SuperScript II reverse transcriptase (Invitrogen). PCR for CD45 and GAPDH were done according to previous Mice report (32). To analyze CD45 alternative splicing, an established radio- C57BL/6, LckCre transgenic, Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo, active RT-PCR protocol was used (33). Briefly, 0.5 mg purified RNA of sorted thymic subpopulations was used for reverse transcription with a and CD45.1 mice were purchased from Jackson ImmunoResearch Labo- 32 ratories or were maintained at the Institut de Recherches Cliniques de gene-specific primer, and PCR was performed with a P-labeled forward Downloaded from Montreal.