Nuclear speckle related protein 70 regulates alternative splicing via Arginine/Serine-like region and thymocyte development

Abstract

Nuclear speckles are a sub-nuclear storage site containing pre-mRNA splicing machinery. Proteins assembled in nuclear speckles are known to modulate transcription and pre-mRNA processing. We have previously identified the nuclear speckle-related protein 70 (NSrp70) as a novel serine/arginine (SR)-related protein that co-localizes with classical SR proteins such as Serine/Arginine-Rich Splicing Factor-1 (SRSF1 or ASF/SF2) and SRSF2 (SC35). NSrp70 mediates alternative splice site selection, targeting several pre-mRNAs including CD44 exon v5. In part I, I demonstrated that NSrp70 physically interacts with two SR proteins, SRSF1 and SRSF2, and reverses their splicing activity in terms of CD44 exon v5 as exon exclusion. The NSrp70 RS-like region was sub-divided into three areas; deletion of the first arginine/serine-rich-like region (RS1) completely abrogated binding to the SR proteins as well as to target mRNA, and also failed to induce splicing of CD44 exon v5, suggesting that RS1 is critical for NSrp70 functioning. Interestingly, RS1 deletion also resulted in the loss of NSrp70 and SR protein speckle positioning, implying a potential scaffolding role for NSrp70 in nuclear speckles. NSrp70 contains an N-terminal coiled-coil (CC) domain that is critical not only for selfoligomerization but also for splicing activity. Consistently, deletion of the CC domain resulted in indefinite formation of nuclear speckles. Collectively, these results demonstrate that NSrp70 acts as a new molecular counterpart for alternative splicing of target RNA, counteracting SRSF1 and SRSF2 splicing activity. In part II, alternative pre-mRNA splicing is a critical step to generate multiple transcripts, thereby dramatically enlarging Thus, a common feature of most alternative splicing factor knockout models is lethality. However, little is known about lineage-specific alternative splicing regulators in a physiological setting. Here, we report that NSrp70 is selectively expressed in developing thymocytes, highest at the double-positive (DP) stage. Global gene profiling revealed that NSrp70 controls cell cycle arrest and survival. A conditional-knockout of Nsrp1 (NSrp70-cKO) using CD4Cre developed severe defects in T cell maturation to single-positive thymocytes, due to insufficient T cell receptor (TCR) signaling and uncontrolled cell growth and death. Mice displayed severe peripheral lymphopenia and could not optimally control tumor growth. This study establishes a model to address the function of lymphoid-lineage-specific alternative splicing factor NSrp70 in a thymic T cell developmental pathway.