T cells transformed by Herpesvirus saimiri express seven viral U-rich noncoding RNAs of unknown function called HSURs. We noted that conserved sequences in HSURs 1 and 2 constitute potential binding sites for three host-cell microRNAs (miRNAs). Coimmunoprecipitation experiments confirmed that HSURs 1 and 2 interact with the predicted miRNAs in virally transformed T cells. The abundance of one of these miRNAs, miR-27, is dramatically lowered in transformed cells, with consequent effects on the expression of miR-27 target genes. Transient knockdown and ectopic expression of HSUR 1 demonstrate that it directs degradation of mature miR-27 in a sequence-specific and binding-dependent manner. This viral strategy illustrates use of a ncRNA to manipulate host-cell gene expression via the miRNA pathway.

Splicing factors of the SR protein family share a modular structure consisting of one or two RNA recognition motifs (RRMs) and a C-terminal RS domain rich in arginine and serine residues. The RS domain, which is extensively phosphorylated, promotes protein-protein interactions and directs subcellular localization and-in certain situations-nucleocytoplasmic shuttling of individual SR proteins. We analyzed mutant versions of human SF2/ASF in which the natural RS repeats were replaced by RD or RE repeats and compared the splicing and subcellular localization properties of these proteins to those of SF2/ASF lacking the entire RS domain or possessing a minimal RS domain consisting of 10 consecutive RS dipeptides (RS10). In vitro splicing of a pre-mRNA that requires an RS domain could take place when the mutant RD, RE, or RS10 domain replaced the natural domain. The RS10 version of SF2/ASF shuttled between the nucleus and the cytoplasm in the same manner as the wild-type protein, suggesting that a tract of consecutive RS dipeptides, in conjunction with the RRMs of SF2/ASF, is necessary and sufficient to direct nucleocytoplasmic shuttling. However, the SR protein SC35 has two long stretches of RS repeats, yet it is not a shuttling protein. We demonstrate the presence of a dominant nuclear retention signal in the RS domain of SC35.Pre-mRNA splicing takes place within the spliceosome, a macromolecular complex composed of four small nuclear ribonucleoprotein particles (snRNPs) (U1, U2, U4/U6, and U5) and approximately 50 to 100 polypeptides (32). The SR proteins are an extensively characterized family of structurally and functionally related non-snRNP splicing factors that are essential for constitutive splicing and that also influence alternative splicing regulation in higher eukaryotes (5,19,58).The SR proteins are involved in multiple steps of the constitutive splicing reaction. They promote the recruitment of the U1 snRNP to the 5Ј splice site (14,26,29) and of the U2AF splicing factor and U2 snRNP to the branch site or 3Ј splice site (57, 62) and also facilitate the recruitment of the U4/U6-U5 tri-snRNP at later stages of the splicing reaction (51). The SR proteins also bridge pairs of 5Ј and 3Ј splice sites via RS domain-mediated protein-protein interactions, which can occur across an exon or an intron (exon definition or intron definition) (50, 62). The SR proteins are also important regulators of alternative splicing, and their activity is subject to antagonism by members of the hnRNP A/B family of proteins. Increased levels of SR proteins usually promote the selection of proximal alternative 5Ј splice sites both in vitro and in vivo, whereas an excess of hnRNPs A/B proteins usually results in the selection of distal 5Ј splice sites (8,38,63). Tissue-specific variations in the total and relative amounts of SR proteins and in the molar ratio of SF2/ASF to its antagonist, hnRNP A1, support the notion that the relative levels and activities of these two families of antagonistic factors may be crucial in regulating th...

Cellular senescence is a form of irreversible growth arrest and a major tumor suppressor mechanism. We show here that the miR-29 and miR-30 microRNA families are up-regulated during induced and replicative senescence and that up-regulation requires activation of the Rb pathway. Expression of a reporter construct containing the 3′UTR of the B-Myb oncogene is repressed during senescence, and repression is blocked by mutations in conserved miR-29 and miR-30 binding sites in the B-Myb 3′UTR. In proliferating cells, transfection of miR-29 and miR-30 represses a reporter construct containing the wild-type but not the mutant B-Myb 3′UTR, and repression of the mutant 3′UTR is reinstituted by compensatory mutations in miR-29 and miR-30 that restore binding to the mutant sites. miR-29 and miR-30 introduction also represses expression of endogenous B-Myb and inhibits cellular DNA synthesis. Finally, interference with miR-29 and miR-30 expression inhibits senescence. These findings demonstrate that miR-29 and miR-30 regulate B-Myb expression by binding to its 3′UTR and suggest that these microRNAs play an important role in Rb-driven cellular senescence.

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...