Role of pre-rRNA base pairing and 80S complex formation in subnucleolar localization of the U3 snoRNP

In the nucleolus the U3 snoRNA is recruited to the 80S pre-rRNA processing complex in the dense fibrillar component (DFC). The U3 snoRNA is found throughout the nucleolus and has been proposed to move with the preribosomes to the granular component (GC). In contrast, the localization of other RNAs, such as the U8 snoRNA, is restricted to the DFC. Here we show that the incorporation of the U3 snoRNA into the 80S processing complex is not dependent on pre-rRNA base pairing sequences but requires the B/C motif, a U3-specific protein-binding element. We also show that the binding of Mpp10 to the 80S U3 complex is dependent on sequences within the U3 snoRNA that base pair with the pre-rRNA adjacent to the initial cleavage site. Furthermore, mutations that inhibit 80S complex formation and/or the association of Mpp10 result in retention of the U3 snoRNA in the DFC. From this we propose that the GC localization of the U3 snoRNA is a direct result of its active involvement in the initial steps of ribosome biogenesis.     

The branchpoint residue is recognized during commitment complex formation before being bulged out of the U2 snRNA-pre-mRNA duplex.

We have analyzed the mechanism of branchpoint nucleotide selection during the first step of pre-mRNA splicing. It has previously been proposed that the branchpoint is selected as an adenosine residue bulged out of an RNA helix formed by the U2 snRNA-pre-mRNA base pairing. Although compatible with this bulge hypothesis, available data from both yeast and mammalian systems did not rule out alternative structures for the branch nucleotide. Mutating the residue preceding the branchpoint nucleotide in our reporter construct conferred a splicing defect that was suppressed in vivo by the complementary U2 snRNA mutants. In contrast, substitutions on the 3' side of the branchpoint could be suppressed by complementary U2 snRNA mutants only in a weakened intron context. To test why the identity of the branch nucleotide was important for its selection, we analyzed the effect of substitutions at this position on spliceosome assembly. We observed that these mutations block the formation of one of the two commitment complexes. Our results demonstrate that yeast branchpoint selection occurs in multiple steps. The nature of the branch residue is recognized, in the absence of U2 snRNA, during commitment complex formation. Then, base pairing with U2 snRNA constrains this residue into a bulge conformation.     

The yeast branchpoint sequence is not required for the formation of a stable U1 snRNA-pre-mRNA complex and is recognized in the absence of U2 snRNA.

Commitment complexes contain U1 snRNP as well as pre-mRNA and are the earliest functional complexes that have been described during in vitro spliceosome assembly. We have used a gel retardation assay to analyze the role of the yeast pre-mRNA cis-acting sequences in commitment complex formation. The results suggest that only a proper 5' splice site sequence is required for efficient U1 snRNA-pre-mRNA complex formation. A role for the highly conserved UACUAAC branchpoint sequence is indicated, however, by competition experiments and by the direct analysis of branchpoint mutant substrates, which cannot form one of the two commitment complex species observed with wild-type substrates. The results suggest that the formation of a U1 snRNP-pre-mRNA complex is not dependent upon the presence of a branchpoint sequence but that the branchpoint sequence is recognized prior to U2 snRNP addition during in vitro spliceosome assembly.     

G.U base pairing motifs in ribosomal RNA.

An increasing number of recognition mechanisms in RNA are found to involve G.U base pairs. In order to detect new functional sites of this type, we exhaustively analyzed the sequence alignments and secondary structures of eubacterial and chloroplast 16S and 23S rRNA, seeking positions with high levels of G.U pairs. Approximately 120 such sites were identified and classified according to their secondary structure and sequence environment. Overall biases in the distribution of G.U pairs are consistent with previously proposed structural rules: the side of the wobble pair that is subject to a loss of stacking is preferentially exposed to a secondary structure loop, where stacking is not as essential as in helical regions. However, multiple sites violate these rules and display highly conserved G.U pairs in orientations that could cause severe stacking problems. In addition, three motifs displaying a conserved G.U pair in a specific sequence/structure environment occur at an unusually high frequency. These motifs, of which two had not been reported before, involve sequences 5''UG3'' 3''GA5'' and 5''UG3'' 3''GU5'', as well as G.U pairs flanked by a bulge loop 3'' of U. The possible structures and functions of these recurrent motifs are discussed.     

Identification of functional U1 snRNA-pre-mRNA complexes committed to spliceosome assembly and splicing

Although both U1 and U2 snRNPs have been implicated in the splicing process, their respective roles in the earliest stages of intron recognition and spliceosome assembly are uncertain. To address this issue, we developed a new strategy to prepare snRNP-depleted splicing extracts using Saccharomyces cerevisiae cells conditionally expressing U1 or U2 snRNP. Complementation analyses and chase experiments show that a stable complex, committed to the splicing pathway, forms in the absence of U2 snRNP. U1 snRNP and a substrate containing both a 5' splice site and a branchpoint sequence are required for optimal formation of this commitment complex. We developed new gel electrophoresis conditions to identify these committed complexes and to show that they contain U1 snRNA. Chase experiments demonstrated that these complexes are functional intermediates in spliceosome assembly and splicing. Our results have implications for the process of splice site selection.     

Evidence for the existence of snRNAs U4 and U6 in a single ribonucleoprotein complex and for their association by intermolecular base pairing

Small nuclear ribonucleoprotein particles (snRNPs) from eucaryotic cells can be fractionated on affinity columns prepared with antibodies of high affinity for 2,2,7-trimethyl-guanosine (m3G), which is present in the 5'-terminal caps of the snRNAs. While the snRNPs U1, U2 and U5 are eluted with the nucleoside m3G in the presence of 0.1 M salt, the snRNP species U4 and U6 are only desorbed when the salt concentration is increased. The same fractionation pattern was likewise observed for snRNPs from HeLa or Ehrlich ascites tumor cells. Since U6 RNA lacks the m3G residue and its RNA does not react with anti-m3G, its co-chromatography with U4 RNP on anti-m3G affinity columns suggests either that discrete snRNPs U4 and U6 are intimately associated in nuclear extracts or that both RNAs are organized in one ribonucleoprotein particle. Further evidence for a U4/U6 RNP particle is obtained by sedimentation studies with purified snRNPs in sucrose gradients. Gel fractionation of RNAs shows identical distributions of snRNAs U4 and U6 in the gradient, and the U4/U6 RNP particle sediments faster than the snRNPs U1 or U2. Physical association between snRNPs U4 and U6 during sedimentation is shown by their co-precipitation with anti-m3G IgG from the gradient fractions. Finally, experimental evidence is provided that snRNAs U4 and U6 are associated by intermolecular base pairing in the U4/U6 RNP particle, as demonstrated by our finding that anti-m3G IgG co-precipitates U6 RNA with U4 RNA following phenolization of U4/U6 RNPs at 0 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Commitment to splice site pairing coincides with A complex formation

Differential recognition of exons by the spliceosome regulates gene expression and exponentially increases the complexity of metazoan proteomes. After definition of the exons, the spliceosome is activated by a series of sequential structural rearrangements. Formation of the first ATP-independent spliceosomal complex commits the pre-mRNA to the general splicing pathway. However, the time at which a commitment to a specific splice site choice and pairing is made is unknown. Here, we demonstrate that alternative splicing patterns are irreversibly chosen at a kinetic step different from the ATP-independent commitment to splicing. Splice sites become committed at the first ATP-dependent spliceosomal complex when rearrangements lock U2 snRNP onto the pre-mRNA. Thus, commitment to the splicing pathway and commitment to splice site pairing are separate steps during spliceosomal assembly, and ATP hydrolysis drives the irreversible juxtaposition of exons within the spliceosome.     

rRNA-mRNA base pairing stimulates a programmed -1 ribosomal frameshift.

Base pairing between the 3' end of 16S rRNA and mRNA is shown to be important for the programmed -1 frameshifting utilized in decoding the Escherichia coli dnaX gene. This pairing is the same as the Shine-Dalgarno pairing used by prokaryotic ribosomes in selection of translation initiators, but for frameshifting the interaction occurs within elongating ribosomes. For dnaX -1 frameshifting, the 3' base of the Shine-Dalgarno sequence is 10 nucleotides 5' of the shift site. Previously, Shine-Dalgarno rRNA-mRNA pairing was shown to stimulate the +1 frameshifting necessary for decoding the release factor 2 gene. However, in the release factor 2 gene, the Shine-Dalgarno sequence is located 3 nucleotides 5' of the shift site. When the Shine-Dalgarno sequence is moved to the same position relative to the dnaX shift site, it is inhibitory rather than stimulatory. Shine-Dalgarno interactions by elongating ribosomes are likely to be used in stimulating -1 frameshifting in the decoding of a variety of genes.     

Adenine-guanine base pairing ribosomal RNA.

Analyses of secondary structures proposed for ribosomal RNA's show that, of the different kinds of base pairs directly adjoining the ends of postulated double-helical regions, only A-G with A at the 5' end significantly exceeds the number expected for a random base distribution. An A(syn)-G(trans) hydrogen-bonded basepair is proposed. This could fit at the end of an undistorted double helix, but would prevent further base stacking, thus favoring a break in the double helix to produce a non-linear tertiary structure.     

The U1 snRNP base pairs with the 5' splice site within a penta-snRNP complex

Recognition of the 5' splice site is an important step in mRNA splicing. To examine whether U1 approaches the 5' splice site as a solitary snRNP or as part of a multi-snRNP complex, we used a simplified in vitro system in which a short RNA containing the 5' splice site sequence served as a substrate in a binding reaction. This system allowed us to study the interactions of the snRNPs with the 5' splice site without the effect of other cis-regulatory elements of precursor mRNA. We found that in HeLa cell nuclear extracts, five spliceosomal snRNPs form a complex that specifically binds the 5' splice site through base pairing with the 5' end of U1. This system can accommodate RNA-RNA rearrangements in which U5 replaces U1 binding to the 5' splice site, a process that occurs naturally during the splicing reaction. The complex in which U1 and the 5' splice site are base paired sediments in the 200S fraction of a glycerol gradient together with all five spliceosomal snRNPs. This fraction is functional in mRNA spliceosome assembly when supplemented with soluble nuclear proteins. The results argue that U1 can bind the 5' splice site in a mammalian preassembled penta-snRNP complex.     

Accessibility of U1 RNA to base pairing with a single-stranded DNA fragment mimicking the intron extremities at the splice junction.

A DNA fragment containing a 16 nucleotide sequence mimicking the intron extremities of premessenger RNA aligned as proposed previously (1,2) in a model of splicing mechanism was prepared and used as a probe for accessibility of the 5' extremity of U1 RNA. Hybridization of U1 RNA to the probe under non denaturing conditions and digestion of the hybrid with RNase H revealed that the sequence of U1 RNA which is complementary to the extremities of introns is accessible to hybridization and to enzymes. Therefore, the configuration of isolated U1 RNA satisfies the criteria required for the alignment of introns and further enzymatic reactions of splicing.     

Parallel stranded DNA with AT base pairing

The concentration and temperature dependences of the UV and CD spectra of the oligonucleotide 3'-d(ApTpApTpApTpApTpApTp)-O(CH2)6O-5'-d(pApTpApTpApTpApT pApT) (eicosamer) in aqueous solution at pH 7 in the presence of 0.5 M NaCl were studied. At less than 10(-6) M, the eicosamer was shown to form in solution a hairpin with parallel orientation of chains (parallel hairpin). From thermal denaturation profiles [A260(T)] the thermodynamic parameters, delta H degrees, delta S degrees and Tm for parallel hairpin formation were calculated to be -90 +/- 8 kJ/mol. -300 +/- 20 J.mol-1.K-1 and 40.5 degrees C, respectively. The CD spectra of the parallel double helix differed from those of B-form DNA and had characteristic features: decreasing magnitude of the positive maximum at 265 nm and a negative peak at 285 nm.     

Mutation as an error in base pairing

Summary The model of mutation by transitional change (Freese 1959) predicts that a heritable change in genotype is established when two replications of DNA succeed the initial incorporation of an analogue. The model was tested in populations ofSalmonella typhimurium strainstryD-10 andtryD-79 whose division had been synchronized by fractional filtration. Mutation from auxotrophy to prototrophy (try ^–try ⁺) induced by 5-bromodeoxyuridine (BUDR) and 2-aminopurine (AP) occurred in accordance with DNA replication. Two subsequent DNA replications were necessary to obtain BUDR-induced prototrophs inD-79, one subsequent DNA replication was required for AP-induced prototrophs inD-79, while no subsequent DNA replication was necessary for AP-induced prototrophs inD-10. This was observed whether the mutagens were present continuously or during only the first replication and also when the cells were allowed to replicate their DNA without cell division in the presence of inhibitory concentrations of the base analogue or when protein synthesis was blocked in the presence of chloramphenicol. A statistical analysis of the patterns of mutant increase observed for six mutant strains was used to distinguish between errors in replication and errors in incorporation induced by the base analogues and thereby the base pair at the mutant site was identified.With 10 Figures in the TextSupported in part by grants from the American Cancer Society the U.S. Public Health Service and the National Science Foundation administered by ProfessorF. J. Ryan.     

Model systems for understanding DNA base pairing

The fact that nucleic acid bases recognize each other to form pairs is a canonical part of the dogma of biology. However, they do not recognize each other well enough in water to account for the selectivity and efficiency that is needed in the transmission of biological information through a cell. Thus proteins assist in this recognition in multiple ways, and recent data suggest that these mechanisms of recognition can vary widely with context. To probe how the chemical differences of the four nucleobases are defined in various biological contexts, chemists and biochemists have developed modified versions that differ in their polarity, shape, size, and functional groups. This brief review covers recent advances in this field of research.