Phosphorylation-dependent Interaction between the Splicing Factors SAP155 and NIPP1

Abstract

NIPP1 is a ubiquitously expressed nuclear protein that functions both as a regulator of protein Ser/Thr phosphatase-1 and as a splicing factor. The N-terminal part of NIPP1 consists of a phosphothreonine-interacting Forkhead-associated (FHA) domain. We show here that the FHA domain of NIPP1 interacts in vitro and in vivo with a TP dipeptide-rich fragment of the splicing factor SAP155/SF3b 155 , a component of the U2 small nuclear ribonucleoprotein particle. The NIPP1-SAP155 interaction was entirely dependent on the phosphorylation of specific TP motifs in SAP155. Mutagenesis and competition studies revealed that various phosphorylated TP motifs competed for binding to the same site in the FHA domain. The SAP155 kinases in cell lysates were blocked by the Ca 2؉ chelator EGTA and by the cyclin-dependent protein kinase inhibitor roscovitine. The phosphorylation level of SAP155 was dramatically increased during mitosis, and accordingly the activity of SAP155 kinases was augmented in mitotic lysates. We discuss how the interaction between NIPP1 and SAP155 could contribute to spliceosome (dis)assembly and the catalytic steps of splicing.Spliceosomes catalyze the removal of intronic sequences from primary transcripts (pre-mRNAs) in two consecutive transesterification reactions (1-3). Their major components are the U1, U2, U5, and U4/U6 small nuclear ribonucleoprotein particles (snRNPs), 1 which consist of small nuclear RNAs and a set of snRNP proteins. These snRNPs are recruited from nuclear storage/assembly sites known as the splicing factor compartments or speckles, and their assembly on pre-mRNAs occurs in a stepwise manner. According to a recent model of spliceosome assembly (2), U1 snRNP and U4/U6⅐U5 tri-snRNP first contact the 5Ј splice site. Subsequently U2 snRNP binds stably to the intronic branch site region. The creation of splicing-competent spliceosomes also depends on the recruitment of numerous nonsnRNP-associated splicing factors and requires multiple, ordered rearrangements between spliceosomal components.Substantial evidence implicates reversible protein phosphorylation in both spliceosome (dis)assembly and splicing catalysis (2, 4). For example, the SR family of splicing factors contain a domain that is rich in Arg/Ser dipeptides, and the phosphorylation of these motifs by specific "SR" kinases has been shown to modulate the interaction of SR proteins with other splicing factors and with RNA during spliceosome assembly (5). The shuttling of SR proteins between the speckles and the spliceosomes and the dispersion of SR proteins from the speckles during mitosis is also controlled by phosphorylation (6 -8). The splicing factor SF1/BBP, which interacts with U2AF65 in an early step of spliceosome assembly, is a substrate for phosphorylation by protein kinase G (9). The phosphorylation of SF1/ BBP inhibits its interaction with U2AF65 and blocks splicing complex formation. A role for protein phosphatases in pre-mRNA splicing has also been firmly established. The protein Ser/Thr phosphatase PP2C␥ is required ...

The transcriptional repressor NIPP1 is an essential player in EZH2-mediated gene silencing

“…NIPP1 binds in vivo to protein Ser/Thr kinase MELK (Vulsteke et al, 2004) and protein Ser/Thr phosphatase-1 , but the physiological implication of these interactions is not known. In addition, NIPP1 interacts with RNA and the essential pre-mRNA splicing factors CDC5L and SAP155 (Jagiello et al, 1997;Jin et al, 1999;Boudrez et al, 2000Boudrez et al, , 2002. Consistent with the latter findings, NIPP1 was reported to be associated with nuclear storage sites of splicing factors as well as with spliceosomes, the RNA-protein complexes that catalyse pre-mRNA splicing .…”

“…Our previous studies identified NIPP1 as a premRNA splicing factor that interacts in vivo with the splicing factors Cdc5L and SAP155, and is needed for a late step of spliceosome assembly (Boudrez et al, , 2002Beullens and Bollen, 2002). We currently do not know whether the splicing function of NIPP1 is somehow linked to its transcriptional repressor function.…”

“…For example, the NIPP1 ligand and U2 snRNP component SAP155 was recently reported to be required for PcG-mediated gene silencing and to interact with PRC1 components (Isono et al, 2005). Since NIPP1 interacts with PRC2 components (Jin et al, 2003;Roy et al, 2007) as well as with phosphorylated forms of SAP155 (Boudrez et al, 2002), it is tempting to speculate that NIPP1 contributes to interactions between PRC1 and PRC2.…”

Unmasking the U2AF homology motif family: a bona fide protein–protein interaction motif in disguise

“…While it has been shown that SF3b155 is successively phosphorylated and dephosphorylated during spliceosome assembly (Wang et al 1998;Boudrez et al 2002;Shi et al 2006), a direct link to the ULM-proximal TP motifs has yet to be established. The SF3b155 subunit is phosphorylated in vitro by cyclin B-cdk1 and cyclin E-cdk2 Boudrez et al 2002), which also co-immunoprecipitate with SF3b155 from cell lysates, and SF3b155 phosphorylation significantly increases during mitosis ). However, various TP motifs in the SF3b155 IDR are predicted by NetPhosK (Blom et al 2004) to be phosphorylated by at least ten different kinases.…”

Inhibition of Spliceosome Assembly by the Cell Cycle-regulated Protein Kinase MELK and Involvement of Splicing Factor NIPP1

“…The FHA domain of NIPP1 mediates targeting to both the spliceosomes and the nuclear storage sites for splicing factors, known as "speckles" (8,10). The targeting function of the FHA domain of NIPP1 is likely explained by its ability to bind to phosphorylated forms of the essential splicing factors CDC5L (11) and SAP155 (12).…”