RNA-dependent phosphorylation of a nuclear RNA binding protein.

The human C1 heterogeneous nuclear ribonucleoprotein particle protein (hnRNP protein) undergoes a cycle of phosphorylation-dephosphorylation in HeLa cell nuclear extracts that modulates the binding of this protein to pre-mRNA. We now report that hyperphosphorylation of the C1 hnRNP protein is mediated by a kinase activity in nuclear extracts that is RNA-dependent. Although the basal phosphorylation of the C1 hnRNP protein in nuclear extracts reflects a casein kinase II-type activity, its RNA-dependent hyperphosphorylation appears to be mediated by a different kinase. This is indicated by the unresponsiveness of the RNA-stimulated hyperphosphorylation to casein kinase II inhibitors, and the distinct glycerol gradient sedimentation profiles of the basal versus RNA-stimulated C1 hnRNP protein phosphorylation activities from nuclear extracts. RNA-dependent phosphorylation was observed both for a histidine-tagged recombinant human C1 hnRNP protein added to nuclear extracts and also for the endogenous C1 hnRNP protein. Additional results rule out protein kinase A, protein kinase C, calmodulin-dependent protein kinase II, and double-stranded RNA-activated protein kinase as the enzymes responsible for the RNA-dependent hyperphosphorylation of the C1 hnRNP protein. These results reveal the existence in nuclear extracts of an RNA-dependent protein kinase activity that hyperphosphorylates a known pre-mRNA binding protein, and define an additional element to be integrated into the current picture of how nuclear proteins are regulated by phosphorylation.

A discrete 3' region of U6 small nuclear RNA modulates the phosphorylation cycle of the C1 heterogeneous nuclear ribonucleoprotein particle protein.

The C heterogeneous ribonucleoprotein particle (hnRNP) protein bind to nascent pre-mRNA and may participate in assembly of the early prespliceosome. Ser/Thr phosphorylation of the C1 hnRNP protein in HeLa nuclear extracts regulates its binding to pre-mRNA (S. H. Mayrand, P. Dwen, and T. Pederson, Proc. Natl. Acad. Sci. USA 90:7764-7768, 1993). We have now further investigated the phosphorylation cycle of the C1 hnRNP protein, with emphasis on its regulation. Pretreatment of nuclear extracts with micrococcal nuclease eliminated the phosphorylation of C1 hnRNP protein, but pretreatment with DNase did not, suggesting a dependence on RNA. Oligodeoxynucleotide-targeted RNase H cleavage of U1, U2, and U4 small nuclear RNAs did not affect the phosphorylation of C1 hnRNP protein. However, cleavage of nucleotides 78 to 95, but not other regions, of U6 small nuclear RNA resulted in an inhibition of the dephosphorylation step of the C1 hnRNP protein phosphorylation cycle. This inhibition was as pronounced as that seen with the serine/threonine protein phosphatase inhibitor okadaic acid. C1 hnRNP protein dephosphorylation could be completely restored by the addition of intact U6 RNA. Add-back experiments with mutant RNAs further delineated the minimal region essential for C1 protein dephosphorylation as residing in nucleotides 85 to 92 of U6 RNA. These results illuminate a hitherto unanticipated function of U6 RNA: the modulation of a phosphorylation-dephosphorylation cycle of C1 hnRNP protein that influences the binding affinity of this protein for pre-mRNA. This newly revealed function of U6 RNA is likely to play a very early role in the prespliceosome assembly pathway, prior to U6 RNA's entry into the mature spliceosome's active center.

Requirement of a small cytoplasmic RNA for the establishment of thermotolerance.

Thermotolerance is an inducible state that endows cells with an enhanced resistance to thermal killing. Heat shock proteins are believed, and in a few instances have been shown, to be the agents conferring this resistance. The role of a small cytoplasmic RNA (G8 RNA) in developing thermotolerance in Tetrahymena thermophila was investigated by creating a strain devoid of all functional G8 genes. These G8 null cells mounted an apparently normal heat shock response, but they were unable to establish thermotolerance.