Temporal regulation of RNA polymerase II by Srb10 and Kin28 cyclin-dependent kinases.

Two cyclin-dependent kinases have been identified in yeast and mammalian RNA polymerase II transcription initiation complexes. We find that the two yeast kinases are indistinguishable in their ability to phosphorylate the RNA polymerase II CTD, and yet in living cells one kinase is a positive regulator and the other a negative regulator. This paradox is resolved by the observation that the negative regulator, Srb10, is uniquely capable of phosphorylating the CTD prior to formation of the initiation complex on promoter DNA, with consequent inhibition of transcription. In contrast, the TFIIH kinase phosphorylates the CTD only after the transcription apparatus is associated with promoter DNA. These results reveal that the timing of CTD phosphorylation can account for the positive and negative functions of the two kinases and provide a model for Srb10-dependent repression of genes involved in cell type specificity, meiosis, and sugar utilization.

AU-rich elements target small nuclear RNAs as well as mRNAs for rapid degradation.

AU-rich elements (AREs, usually containing repeated copies of AUUUA), when present in the 3'-untranslated regions (UTRs) of many mammalian mRNAs, confer instability on their host RNA molecules. The viral small nuclear RNA (snRNA) Herpesvirus saimiri U RNA 1 (HSUR 1) also contains an AUUUA-rich sequence. Here, we report that this ARE induces rapid degradation of HSUR 1 itself and of other snRNAs including HSUR 2 and cellular U1. Mutational analyses of the viral ARE establish that sequence requirements for mRNA and snRNA decay are the same, suggesting a similar mechanism. Moreover, the in vivo degradation activity of mutant AREs correlates with their in vitro binding to the HuR protein, implicated previously as a component of the mRNA degradation machinery. Our results suggest that ARE-mediated instability can be uncoupled from both ongoing translation and deadenylation of the target RNA.

Identification of HuR as a protein implicated in AUUUA-mediated mRNA decay.

Expression of many proto-oncogenes, cytokines and lymphokines is regulated by targeting their messenger RNAs for rapid degradation. Essential signals for this control are AU-rich elements (AREs) in the 3' untranslated region (UTR) of these messages. The ARE is loosely defined as the five-nucleotide sequence AUUUA embedded in a uracil-rich region. A transacting factor, presumably a protein, binds the ARE and initiates recognition by the destabilization machinery. Numerous candidate ARE-binding proteins have been proposed. We show that a 32 kDa protein in HeLa nuclear extracts characterized previously has RNA-binding specificity that correlates with the activity of an ARE in directing mRNA decay. Purification and subsequent analyses demonstrate that this 32 kDa protein is identical to a recently identified member of the Elav-like gene family (ELG) called HuR. The in vitro binding selectivity of HuR is indicative of an ARE sequence's ability to destabilize a mRNA in vivo, suggesting a critical role for HuR in the regulation of mRNA degradation.

Isolation and characterization of a novel, low abundance hnRNP protein: A0.

Pre-messenger RNA is bound by a variety of proteins to form large heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. As defined by immunoprecipitation and two-dimensional gel electrophoresis, there appear to be more than 20 abundant hnRNP proteins ranging in size from 34 kDa to 120 kDa. One major class, the A/B family, is typified by its characteristic primary structure containing two RNA binding domains followed by a glycine-rich C-terminus. We report the cloning and characterization of a novel, low-abundance member of the A/B family named hnRNP A0. This protein was affinity isolated using a biotinylated RNA probe [G4(AU3)4A] designed to select a 32-kDa protein implicated in mRNA stability in mammalian cells. hnRNP A0 is a basic protein with a predicted mass of 31.7 kDa and an isoelectric point of 10.1. Comparative protease mapping shows that it is not the AUUUA binding protein we intended to clone. A0 is present in hnRNP complexes and is encoded by a gene distinct from that of any previously cloned A/B family member.

Viral small nuclear ribonucleoproteins bind a protein implicated in messenger RNA destabilization.

Herpesvirus saimiri (HVS) is one of several primate viruses that carry genes for small RNAs. The five H. saimiri-encoded U RNAs (HSURs) are the most abundant viral transcripts expressed in transformed marmoset T lymphocytes. They assemble with host proteins common to spliceosomal small nuclear ribonucleoproteins (snRNPs). HSURs 1, 2, and 5 exhibit sequences at their 5' ends identical to the AUUUA motif, which targets a number of protooncogene, cytokine, and lymphokine mRNAs for rapid degradation. We show that a 32-kDa protein previously demonstrated to bind to the 3' untranslated region of several unstable messages can be UV crosslinked specifically to HSUR 1, 2, and 5 transcripts in vitro, as well as to endogenous HSUR snRNPs. Our results suggest an unusual role for these viral snRNPs: HSURs may function to attenuate the rapid degradation of certain cellular mRNAs, thereby facilitating viral transformation of host T lymphocytes.