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.

Serine/threonine phosphorylation regulates binding of C hnRNP proteins to pre-mRNA.

The C hnRNP proteins bind to nascent pre-mRNA and are thought to participate in an early step of the pre-mRNA splicing pathway. We report here that C hnRNP proteins are phosphorylated by a casein kinase II activity in a HeLa cell nuclear extract and that dephosphorylation of C hnRNP proteins is inhibited by the specific protein-serine/threonine-phosphatase 1/2A inhibitor okadaic acid. We further find that dephosphorylation of C hnRNP proteins is required for their binding to adenovirus and human beta-globin pre-mRNAs. These results indicate that the participation of C hnRNP proteins in pre-spliceosome assembly is coupled to a dynamic cycle of their phosphorylation and dephosphorylation.

Crosslinking of hnRNP proteins to pre-mRNA requires U1 and U2 snRNPs.

Proteins interacting with pre-mRNAs during early stages of spliceosome formation in a HeLa nuclear extract were investigated by photochemical RNA-protein crosslinking. The level of protein crosslinking to a beta-globin pre-mRNA was positively correlated with the presence of an intron. Proteins of 110,000, 59,000 and 39,000 mol. wt. were crosslinked to the beta-globin pre-mRNA, the latter of which was identified as the A1 hnRNP protein. Comparable experiments with an adenovirus pre-mRNA revealed crosslinked proteins of 110,000, 56,000 and 45,000 mol. wt., with the latter identified as belonging to the C group hnRNP proteins. Crosslinking of hnRNP proteins to both the beta-globin and adenovirus pre-mRNAs was eliminated by oligodeoxynucleotide-directed RNase H excision of an internal region (nt 28-42) of U2 RNA, but was not affected by oligo/RNase H cleavage of the 5'-terminal 15 nucleotides of U2 RNA. Cleavage of the 5'-terminal 15 nucleotides of U1 RNA preferentially eliminated crosslinking of the hnRNP A1 protein to both pre-mRNAs. The requirement of intact U1 snRNP for A1 protein crosslinking was further demonstrated by the fact that although micrococcal nuclease-treated extracts did not support crosslinking of A1 hnRNP protein to beta-globin pre-mRNA, crosslinking was restored by addition of a U1 snRNP-enriched fraction.

Site-specific excision from RNA by RNase H and mixed-phosphate-backbone oligodeoxynucleotides.

Oligodeoxynucleotides containing phosphodiester or modified internucleoside linkages were investigated with respect to their ability to be acted on by ribonuclease H activities present in a HeLa cell nuclear extract after hybridization with complementary sequences in RNA. Oligodeoxynucleotides complementary to nucleotides 2-14 of human U1 small nuclear RNA were investigated. Extensive cleavage of U1 RNA was observed with the unmodified oligodeoxynucleotide and with the phosphorothioate analogue but not with U1-complementary oligodeoxynucleotides containing methylphosphonate, phosphoro-N-morpholidate, or phosphoro-N-butylamidate internucleoside linkages. Additional experiments using a 514-nucleotide-long RNA substrate demonstrated the capacity of complementary phosphodiester- and phosphorothioate-linked oligodeoxynucleotides (but not ones containing methylphosphonate, phosphoro-N-morpholidate, or phosphoro-N-butylamidate linkages) to serve as RNase H targets when hybridized to an internal RNA site. Detailed comparisons revealed phosphodiester-linked oligodeoxynucleotides to be more efficient than the comparable phosphorothioate-linked oligomers with respect to RNase H action. Various pentadecamer oligodeoxynucleotides complementary to the 514-nucleotide-long test RNA and containing 2-6 consecutive phosphodiester- or phosphorothioate-linked nucleotides flanked by RNase H-resistant methylphosphonate linkages afforded precise "site-directed" RNase H excision within the DNA.RNA hybrid. These results serve to assort modified oligodeoxynucleotide-containing hybrids into RNase H-sensitive and -resistant classes and also provide clues as to how RNase H makes contact with the DNA strand in a DNA.RNA hybrid.

Identification of proteins that bind tightly to pre-mRNA during in vitro splicing.

Incubation of a human beta-globin pre-mRNA in a HeLa cell nuclear extract under conditions permissive for efficient splicing resulted in the assembly of the RNA into ribonucleoprotein (RNP) complexes. This RNP formation occurred largely within the characteristic lag period that precedes splicing. Two classes of RNP were detected by the criterion of their stability in Cs2SO4 gradients. One was unstable and contained mainly aberrant RNA cleavage products. The other class of RNP complexes comprised 50-85% of the beta-globin RNA, formed only under splicing-permissive conditions, was stable in Cs2SO4 gradients, and contained both unspliced pre-mRNA molecules and the lariat intron 1-exon 2 splicing intermediate. This latter class of RNP complexes banded at approximately equal to 1.30 g/cm3, a density very similar to that of native heterogeneous nuclear RNP particles that contain pre-mRNA. RNA-protein crosslinking revealed major proteins of Mr approximately equal to 38,000 and 41,000 in the stable class of RNP. The use of antibodies specific for heterogeneous nuclear RNP core proteins and for small nuclear RNA-associated proteins, in conjunction with [32P]RNA-protein crosslinking, revealed polypeptides having the molecular weights of both sets of antigens. These results show that both heterogeneous nuclear RNP particle core proteins and small nuclear RNA-associated proteins bind tightly to pre-mRNA during splicing in vitro.