A conditional U5 snRNA mutation affecting pre-mRNA splicing and nuclear pre-mRNA retention identifies SSD1/SRK1 as a general splicing mutant suppressor. (33/1744)

A combination of point mutations disrupting both stem 1 and stem 2 of U5 snRNA (U5AI) was found to confer a thermosensitive phenotype in vivo. In a strain expressing U5AI, pre-mRNA splicing was blocked before the first step through an inability of the mutant U5 snRNA to efficiently associate with the U4/U6 di-snRNP. Formation of early splicing complexes was not affected in extracts prepared from U5 snRNA mutant cells, while the capacity of these extracts to splice a pre-mRNA in vitro was greatly diminished. In addition, significant levels of a translation product derived from intron containing pre-mRNAs could be detected in vivo. The SSD1/SRK1 gene was identified as a multi-copy suppressor of the U5AI snRNA mutant. Single copy expression of SSD1/SRK1 was sufficient to suppress the thermosensitive phenotype, and high copy expression partially suppressed the splicing and U4/U6.U5 tri-snRNP assembly pheno-types. SSD1/SRK1 also suppressed thermosensitive mutations in the Prp18p and U1-70K proteins, while inhibiting growth of the cold sensitive U1-4U snRNA mutant at 30 degrees C. Thus we have identified SSD1/SRK1 as a general suppressor of splicing mutants.  (+info)

Base pairing between U3 small nucleolar RNA and the 5' end of 18S rRNA is required for pre-rRNA processing. (34/1744)

The loop of a stem structure close to the 5' end of the 18S rRNA is complementary to the box A region of the U3 small nucleolar RNA (snoRNA). Substitution of the 18S loop nucleotides inhibited pre-rRNA cleavage at site A(1), the 5' end of the 18S rRNA, and at site A(2), located 1.9 kb away in internal transcribed spacer 1. This inhibition was largely suppressed by a compensatory mutation in U3, demonstrating functional base pairing. The U3-pre-rRNA base pairing is incompatible with the structure that forms in the mature 18S rRNA and may prevent premature folding of the pre-rRNA. In the Escherichia coli pre-rRNA the homologous region of the 16S rRNA is also sequestered, in that case by base pairing to the 5' external transcribed spacer (5' ETS). Cleavage at site A(0) in the yeast 5' ETS strictly requires base pairing between U3 and a sequence within the 5' ETS. In contrast, the U3-18S interaction is not required for A(0) cleavage. U3 therefore carries out at least two functionally distinct base pair interactions with the pre-rRNA. The nucleotide at the site of A(1) cleavage was shown to be specified by two distinct signals; one of these is the stem-loop structure within the 18S rRNA. However, in contrast to the efficiency of cleavage, the position of A(1) cleavage is not dependent on the U3-loop interaction. We conclude that the 18S stem-loop structure is recognized at least twice during pre-rRNA processing.  (+info)

Maximization of selenocysteine tRNA and U6 small nuclear RNA transcriptional activation achieved by flexible utilization of a Staf zinc finger. (35/1744)

Transcriptional activators Staf and Oct-1 play critical roles in the activation of small nuclear RNA (snRNA) and snRNA-type gene transcription. Recently, we established that Staf binding to the human U6 snRNA (hU6) and Xenopus selenocysteine tRNA (xtRNA(Sec)) genes requires different sets of the seven C2-H2 zinc fingers. In this work, using a combination of oocyte microinjection, electrophoretic mobility shift assays, and missing nucleoside experiments with wild-type and mutant promoters, we demonstrate that the hU6 gene requires zinc fingers 2-7 for Staf binding and Oct-1 for maximal transcriptional activity. In contrast, the xtRNA(Sec) gene needs the binding of the seven Staf zinc fingers, but not Oct-1, for optimal transcriptional capacity. Mutation in the binding site for Staf zinc finger 1 in the tRNA(Sec) promoter reduced both Staf binding and transcriptional activity. Conversely, introduction of a zinc finger 1 binding site in the hU6 promoter increased Staf binding but interfered with the simultaneous Staf and Oct-1 binding, thus reducing transcriptional activity. Collectively, these results show that the differential utilization of Staf zinc finger 1 represents a new, critical determinant of the transcriptional activation mechanism for the Xenopus tRNA(Sec) and human U6 snRNA genes.  (+info)

Molecular dynamics simulations of the complex between human U1A protein and hairpin II of U1 small nuclear RNA and of free RNA in solution. (36/1744)

RNA-protein interactions are essential to a wide range of biological processes. In this paper, a 0.6-ns molecular dynamics simulation of the sequence-specific interaction of human U1A protein with hairpin II of U1 snRNA in solution, together with a 1.2-ns simulation of the free RNA hairpin, is reported. Compared to the findings in the x-ray structure of the complex, most of the interactions remained stable. The nucleotide U8, one of the seven conserved nucleotides AUUGCAC in the loop region, was unusually flexible during the simulation, leading to a loss of direct contacts with the protein, in contrast to the situation in the x-ray structure. Instead the sugar-phosphate backbone of nucleotide C15 was found to form several interactions with the protein. Compared to the NMR structure of U1A protein complexed with the 3'-untranslated region of its own pre-mRNA, the protein core kept the same conformation, and in the two RNA molecules the conserved AUUGCAC of the loop and the closest CG base pair were located in very similar positions and orientations, and underwent very similar interactions with the protein. Therefore, a common sequence-specific interaction mechanism was suggested for the two RNA substrates to bind to the U1A protein. Conformational analysis of the RNA hairpin showed that the conformational changes of the RNA primarily occurred in the loop region, which is just involved in the sites of binding to the protein and in agreement with experimental observation. Both the loop and stem of the RNA became more ordered upon binding to the protein. It was also demonstrated that the molecular dynamics method could be successfully used to simulate the dynamical behavior of a large RNA-protein complex in aqueous solution, thus opening a path for the exploration of the complex biological processes involving RNA at a molecular level.  (+info)

Spliceosomal U snRNP core assembly: Sm proteins assemble onto an Sm site RNA nonanucleotide in a specific and thermodynamically stable manner. (37/1744)

The association of Sm proteins with U small nuclear RNA (snRNA) requires the single-stranded Sm site (PuAU(4-6)GPu) but also is influenced by nonconserved flanking RNA structural elements. Here we demonstrate that a nonameric Sm site RNA oligonucleotide sufficed for sequence-specific assembly of a minimal core ribonucleoprotein (RNP), which contained all seven Sm proteins. The minimal core RNP displayed several conserved biochemical features of native U snRNP core particles, including a similar morphology in electron micrographs. This minimal system allowed us to study in detail the RNA requirements for Sm protein-Sm site interactions as well as the kinetics of core RNP assembly. In addition to the uridine bases, the 2' hydroxyl moieties were important for stable RNP formation, indicating that both the sugar backbone and the bases are intimately involved in RNA-protein interactions. Moreover, our data imply that an initial phase of core RNP assembly is mediated by a high affinity of the Sm proteins for the single-stranded uridine tract but that the presence of the conserved adenosine (PuAU.) is essential to commit the RNP particle to thermodynamic stability. Comparison of intact U4 and U5 snRNAs with the Sm site oligonucleotide in core RNP assembly revealed that the regions flanking the Sm site within the U snRNAs facilitate the kinetics of core RNP assembly by increasing the rate of Sm protein association and by decreasing the activation energy.  (+info)

Nucleolar factors direct the 2'-O-ribose methylation and pseudouridylation of U6 spliceosomal RNA. (38/1744)

The nucleolus has long been known as a functionally highly specialized subnuclear compartment where synthesis, posttranscriptional modification, and processing of cytoplasmic rRNAs take place. In this study, we demonstrate that the nucleolus contains all the trans-acting factors that are responsible for the accurate and efficient synthesis of the eight 2'-O-methylated nucleotides and three pseudouridine residues carried by the mammalian U6 spliceosomal small nuclear RNA. Factors mediating the formation of pseudouridine residues in the U3 small nucleolar RNA are also present and functionally active in the nucleolus. For selection of the correct target nucleotides in the U6 and U3 RNAs, the nucleolar 2'-O-methylation and pseudouridylation factors rely on short sequences located around the target nucleotide to be modified. This observation further underscores a recently proposed role for small nucleolar guide RNAs in the 2'-O-methylation of the U6 spliceosomal RNA (K. T. Tycowski, Z.-H. You, P. J. Graham, and J. A. Steitz, Mol. Cell 2:629-638, 1998). We demonstrate that a novel 2'-O-methylated nucleotide can be generated in the yeast U6 RNA by use of an artificial 2'-O-methylation small nucleolar guide RNA. We also show that a short fragment of the 5.8S rRNA, when expressed as part of the human U6 RNA, is faithfully 2'-O-methylated and pseudouridylated. These results are most consistent with a trafficking pathway in which the U6 spliceosomal RNA cycles through the nucleolus to undergo nucleolar RNA-directed modifications.  (+info)

Reversion of human glioblastoma malignancy by U1 small nuclear RNA/ribozyme targeting of scatter factor/hepatocyte growth factor and c-met expression. (39/1744)

BACKGROUND: Expression of scatter factor (SF), also known as hepatocyte growth factor (HGF), and its receptor, c-met, is often associated with malignant progression of human tumors, including gliomas. Overexpression of SF/HGF in experimental gliomas enhances tumorigenicity and tumor-associated angiogenesis (i.e., growth of new blood vessels). However, the role of endogenous SF/HGF or c-met expression in the malignant progression of gliomas has not been examined directly. In this study, we tested the hypothesis that human glioblastomas can be SF/HGF-c-met dependent and that a reduction in endogenous SF/HGF or c-met expression can lead to inhibition of tumor growth and tumorigenicity. METHODS: Expression of the SF/HGF and c-met genes was inhibited by transfecting glioblastoma cells with chimeric transgenes consisting of U1 small nuclear RNA, a hammerhead ribozyme, and antisense sequences. The effects of reduced SF/HGF and c-met expression on 1) SF/HGF-dependent induction of immediate early genes (c-fos and c-jun), indicative of signal transduction; 2) anchorage-independent colony formation (clonogenicity), an in vitro correlate of solid tumor malignancy; and 3) intracranial tumor formation in immunodeficient mice were quantified. Statistical tests were two-sided. RESULTS: Introduction of the transgenes into glioblastoma cells reduced expression of the SF/HGF and c-met genes to as little as 2% of control cell levels. Reduction in c-met expression specifically inhibited SF/HGF-dependent signal transduction (P<.01). Inhibition of SF/HGF or c-met expression in glioblastoma cells possessing an SF/HGF-c-met autocrine loop reduced tumor cell clonogenicity (P =.005 for SF/HGF and P=.009 for c-met) and substantially inhibited tumorigenicity (P<.0001) and tumor growth in vivo (P<.0001). CONCLUSIONS: To our knowledge, this is the first successful inhibition of SF/HGF and c-met expression in a tumor model directly demonstrating a role for endogenous SF/HGF and c-met in human glioblastoma. Our results suggest that targeting the SF/HGF-c-met signaling pathway may be an important approach in controlling tumor progression.  (+info)

Identification of an RNA-protein bridge spanning the ribosomal subunit interface. (40/1744)

The 7.8 angstrom crystal structure of the 70S ribosome reveals a discrete double-helical bridge (B4) that projects from the 50S subunit, making contact with the 30S subunit. Preliminary modeling studies localized its contact site, near the bottom of the platform, to the binding site for ribosomal protein S15. Directed hydroxyl radical probing from iron(II) tethered to S15 specifically cleaved nucleotides in the 715 loop of domain II of 23S ribosomal RNA, one of the known sites in 23S ribosomal RNA that are footprinted by the 30S subunit. Reconstitution studies show that protection of the 715 loop, but none of the other 30S-dependent protections, is correlated with the presence of S15 in the 30S subunit. The 715 loop is specifically protected by binding free S15 to 50S subunits. Moreover, the previously determined structure of a homologous stem-loop from U2 small nuclear RNA fits closely to the electron density of the bridge.  (+info)