T cell deficiencies resulting from aberrant pre-mRNA alternative splicing caused by a novel splicing silencer hnRNP LL in an ENU mutant mouse strain thunder
Abstract
ENU mutagenesis screening is a phenotype-driven approach to identify genes in a nonbiased manner. Through this approach we identified a previously uncharacterised gene, Hnrpll, which is involved in nascent mRNA alternative splicing. Thunder strain carries a point mutation in the Hnrpll gene (407T->A) in its first RNA recognition motif (RRM) which changes it function in regulating nascent mRNA alternative splicing.
One of Hnrpll target genes is CD45 that undergoes alternative splicing in T cells depending on their development stages and activation status. We demonstrated that Hnrpllthu/thu T cells fail to silence 3 variable exons of CD45 and result in constitutive expression of CD45RA, B, and C epitopes on the cell surface. Retroviral based expression of wild-type Hnrpll cDNA in the Hnrpllthu/thu T cells compensates the effects of loss of function in the mutation. The mutated RRM1 domain remains the ability to bind the regulatory element of activation responsive sequence (ARS) within CD45 pre-mRNA but cripples the protein function by destabilising the proteins to unfold in a thermolible sensitive manner. We also found that Hnrpllthu/thu mutation disrupts peripheral T cell subsets. Thunder mice have normal T cell development in the thymus but specifically lost naïve T cells in the peripheral lymphoid tissues, whereas memory T cells are not affected. Hnrpllthu/thu naïve T cells can homeostatically proliferate but fail to persist for a long term in vivo, suggesting that thunder mutation influences naïve T cell longevity.
We observed that Hnrpllthu/thu naïve T cells express lower level of IL-7Ra and lower Bcl-2, together with the stronger pro-apoptotic BimS isoform due to alternative splicing. This highlights the nonredundant role of the Hnrpll gene in regulating peripheral T cell homeostasis. CD45 isoforms are widely used markers to distinguish naïve and memory T cell subsets. CD45 splicing does not account for the loss of naïve T cells in the thunder mice, however, CD45RABC shows stronger phosphatase activity than CD45RO when the amount of CD45 is dramatically reduced to 5% remaining on T cell surface, suggesting jointly regulation of CD45 catalytic activity by its expression and alternative splicing. Immunological memory is the hallmark of the adaptive immune system. Through Affymetrix mouse all exon arrays, we found that hnRNP LL protein plays a critical role in controlling an extensive program of alternative splicing as naïve T cells differentiate to the memory cell fate. It acts as an either trans- or cis- acting factor in regulating multiple nascent mRNA alternative splicing. This study provides an unprecedented insight into the extent of alternative splicing in the generation of immune memory.