Nuclear speckles are subnuclear storage sites containing pre-mRNA splicing machinery. Proteins assembled in nuclear speckles are known to modulate transcription and pre-mRNA processing. We have previously identified 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. Here we demonstrated that NSrp70 interacts physically 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 subdivided into three areas. Deletion of the first arginine/serine-rich-like region (RS1) completely abrogated binding to the SR proteins and 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 domain that is critical not only for self-oligomerization but also for splicing activity. Consistently, deletion of the coiled-coil 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.The mammalian cell nucleus contains non-membranous subnuclear bodies such as the nucleolus, Cajal bodies, promyelocytic leukemia bodies, and nuclear speckles. These bodies are generally classified by the presence of a distinct group of proteins and RNAs within them (1, 2). Nuclear speckles are irregular, punctuate structures that vary in size and shape at the immunofluorescence microscope level and are located in interchromatin regions of the nucleoplasm of mammalian cells (3,4). Several reports have demonstrated that nuclear speckles serve as storage/assembly sites for pre-mRNA splicing machinery and, therefore, elaborately regulate gene expression (2). Nuclear speckles are known to be functional centers that spatially organize transcriptional and posttranscriptional mechanisms of gene regulation. RNA-binding proteins involved in transcription and pre-mRNA processing, such as small nuclear ribonucleoprotein and arginine/serine-rich family factors (SR protein, SR-related protein), are associated with nuclear speckles (5, 6). Remarkably, two classical SR proteins, SRSF1 and SRSF2, are well known components of nuclear speckles. These SR proteins modulate both constitutive and alternative splicing by assembly of the mature spliceosome (7,8).In a previous report, we identified a novel SR-related protein, nuclear speckle-related protein 70 (NSrp70), that modulates alternative splicing site selection of several mRNAs in vivo, including Tra21 v2, Fas v6, and...