Reversible phosphorylation differentially affects nuclear and cytoplasmic functions of splicing factor 2/alternative splicing factor

Abstract

The Ser͞Arg-rich (SR) proteins constitute a family of highly conserved nuclear phosphoproteins that are involved in many steps of mRNA metabolism. Previously, we demonstrated that shuttling SR proteins can associate with translating ribosomes and enhance translation of reporter mRNAs both in vivo and in vitro. Here, we show that endogenous, cytoplasmic splicing factor 2͞alternative splicing factor (SF2͞ASF) associated with the translation machinery is hypophosphorylated, suggesting that the phosphorylation state of the Arg-Ser-rich (RS) domain may influence the role of SF2͞ASF in cytoplasmic RNA processing. In agreement, we show that mutations mimicking a hypophosphorylated RS domain strongly increased SF2͞ASF binding to cytoplasmic mRNA and its activity in translation. We also demonstrate that, whereas the RS domain is not required for the function of SF2͞ASF in mRNA translation in vivo or in vitro, its second RNA recognition motif (RRM)2 plays a critical role in this process. Taken together, these data suggest that RS-domain phosphorylation may influence the association of SF2͞ASF with mRNA, whereas RRM2 may play an important role in mediating protein-protein interactions during translation. These data are consistent with a model whereby reversible protein phosphorylation differentially regulates the subcellular localization and activity of shuttling SR proteins.splicing factors ͉ translation ͉ subcellular localization ͉ RNA binding T he Ser͞Arg-rich (SR) proteins are a family of phylogenetically conserved, structurally related, splicing factors that have dual roles in pre-mRNA splicing affecting both constitutive and alternative splicing (1). SR family proteins have a modular structure consisting of one or two copies of an N-terminal RNA-recognition motif (RRM) followed by a C-terminal domain rich in alternating Ser and Arg residues, known as the Arg-Ser-rich (RS) domain. The RRMs determine RNA binding specificity, whereas the RS domain functions as a proteinprotein interaction module by recruiting components of the core splicing apparatus to promote splice site pairing (reviewed in ref.2).SR proteins are primarily localized to the nuclear speckles (reviewed in ref.3), and a subset of SR proteins shuttle continuously between the nucleus and the cytoplasm (4). Within the cell, the RS domain acts as a nuclear localization signal by mediating the interaction with the SR protein nuclear import receptor transportin-SR (5-7) and also influences the nucleocytoplasmic shuttling of individual SR proteins (4). The shuttling ability of a subset of SR proteins suggested additional roles in mRNA transport, and͞or in cytoplasmic events, such as mRNA localization, stability, or regulation of translation. In agreement with this hypothesis, two shuttling SR proteins, SRp20 and 9G8, have been shown to promote mRNA export of intronless RNAs (8) and also act as adapter proteins for TAP-dependent mRNA export (9). SR proteins have also been implicated in RNA stability and quality control. For instance, splicing factor 2͞al-ternati...

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“…Many RBPs such as heterogeneous nuclear RNPs (hnRNPs) and Ser/Arg rich proteins (SR proteins) are associated with mRNA as mRNPs in RNA metabolism, and functionally regulate multiple steps between transcription and translation by dynamic remodeling of RNPs (7). For example, the SR protein that is a genuine alternative splicing factor SF2/ASF acts from alternative splicing to translation (8,9), which suggests a long RNA metabolic journey of a single RBP and its fine tuning by phosphorylation (10). Multiple roles of RBPs would provide a means of interconnecting the post-transcriptional steps, as pro-http://bmbreports.org posed by an extremely simplified model shown in Fig.…”

The SR Protein 9G8 and the Wilms' Tumor Suppressor Protein WT1 Promote Translation of mRNAs with Retained Introns

“…However, unlike 9G8, none of these endogenous proteins are detected in anything larger than the very lightest of the polysomes. This is in contrast to the Sanford studies showing transfected ASF/SF2 in heavy polysomes (175,176). It may be that over-expression of this SR protein drove the protein into larger polysomes than it usually exists (as can happen with 9G8; compare…”

“…21A and 24A). Another possibility is that in our experiment, the ASF protein in these fractions is hypophosphorylated (175), and is, therefore, undetectable by mAb104.…”

“…In this study, it was reported that the SF2/ASF protein was able to promote translation of mRNAs from reporter constructs containing SR protein binding sites, and there was no difference in expression from the reporter mRNA regardless of whether or not it had undergone splicing. Several recent studies have now provided additional support for a role of SR proteins in translation (14,175).…”

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“…The phosphorylation state of the SR domain influences the role of SF2/ASF in cytoplasmic RNA processing. A model has been proposed whereby reversible protein phosphorylation differentially regulates the subcellular localization and activity of shuttling SR proteins [73].…”