Lethal and temperature-sensitive mutations and their suppressors identify an essential structural element in U2 small nuclear RNA.

Abstract

U2 snRNA is an essential component of the splicing apparatus in eukaryotic cells. Three possible secondary structures for the highly conserved 5' half of U2 snRNA are consistent with U2 phylogenetic sequence variation. To distinguish among these models and to test the function of U2 structural elements, we made >35 mutations in the yeast U2 snRNA gene. Some of the mutations were designed in pairs so that combinations could be made that would restore base-pairing to differentiate helix requirements from primary sequence requirements. The mutations identify an essential stem-and-loop structure adjacent to the branchpoint interaction region. A conserved complementarity to the loop just upstream of the Sm site and an additional conserved stem-loop are dispensable for U2 function, even in the background of a previously identified large internal deletion. Non-Watson-Crick base appositions at the 53-62 base pair in the essential stem lead to a variety of temperature and KCl-sensitive phenotypes, as well as an accumulation of unspliced precursors in vivo. Chemical structure probing of U2 RNA in vivo reveals that the bulk of U2 in a yeast cell adopts a structure in good agreement with that deduced from genetic results. We suggest that this stem-loop is not a binding site for an intrinsic U2 snRNP protein but may interact with other factors during spliceosome assembly or splicing.

Sequences upstream of the branch site are required to form helix II between U2 and U6 snRNA in a trans-splicing reaction

“…U2 snRNP appears to exist in two conformations: the familiar conformation required for spliceosome assembly where U2 stem-loop IIa is intact (74), and a phylogenetically conserved alternative conformation where stem-loop IIa is disrupted in order to pair with a sequence immediately downstream from stem-loop IIb (39,74) (Fig. 5B).…”

Inhibition of viral gene expression by human ribonuclease P

“…Since most mRNA species inside cells are usually associated with proteins and are present in a highly organized and folded conformation, it is critical to choose a targeting region that is accessible to binding of EGSs in order to achieve efficient targeting+ In vivo mapping with dimethyl sulphate (DMS) has been extensively used to determine the accessibility of mRNA and structure of RNAs in cells (Climie & Friesen, 1988;Ares & Igel, 1990;Liu & Altman, 1995;Zaug & Cech, 1995)+ Using this method, we mapped the region around the translation initiation site of TK mRNA because this region is supposed to be accessible to ribosome binding (Liu & Altman, 1995)+ A position 29 nt downstream from the TK translational initiation codon was chosen as the cleavage site for human RNase P+ This site appeared to be one of the regions most accessible to DMS modification (data not shown, Liu & Altman, 1995)+ Moreover, its flanking sequence exhibited several sequence features that need to be present in order to interact with an EGS and RNase P to achieve efficient cleavage+ These necessities are that (1) the nucleotides 39 and 59 adjacent to the site of cleavage are a guanosine and a pyrimidine, respectively; and (2) a uracyl is 8 nt downstream from this cleavage site (Yuan & Altman, 1994;Yuan et al+, 1992)+ The interactions of these sequence elements with the EGS facilitate the formation of the mRNA-EGS complex into a tRNA-like structure while FIGURE 1. Schematic representation of substrates for RNase P+ A: Natural substrate (ptRNA)+ B: A hybridized complex of a target RNA (e+g+ mRNA) and an EGS that resembles the structure of a tRNA+ C, D, E: Complexes between TK mRNA sequence and EGS TK104, TK109 and TK112, respectively+ The sequence of these EGSs equivalent to the tRNA sequence (i+e+ T-stem and loop, anticodon stem and loop, and variable region) was derived from tRNA ser + Only the exact sequence of the TK mRNA around the targeting site was shown and italicized+ The EGS sequence was shown in bold type+ The design and construction of these EGSs can be found in Materials and Methods+ those with RNase P are critical for recognition and cleavage by the enzyme (Yuan & Altman, 1994)+ Three EGSs were designed based on the sequence of tRNA ser and constructed (Fig+ 1C-E) as described previously (Yuan & Altman, 1994;Yuan et al+, 1992)+ EGS TK104 (Fig+ 1C) resembles a three-quarters tRNA ser structure (Fig+ 1B) (Yuan et al+, 1992); EGS TK109 (Fig+ 1D) was constructed from TK104 by introducing a point mutation (from C to G) at a highly conserved position in the T-loop+ This cytosine was found in the sequences of all known natural tRNAs (Sprinzl et al+, 1991) and in substrates selected to be efficiently cleaved by human RNase P in simulated evolution in vitro experiments (Yuan & Altman, 1994)+ These observations suggested that this nucleotide may be important for the interaction between the tRNA domains and human RNase P+ F...…”

Complementary deletions in expressed potato U2snRNA gene variants support the hypothesis that stem—loop IIb is dispensable for splicing

“…The underlin and C' represent the stems of stem-loops lined regions B and ontained the somewhat loose plant i exception of the initial C residue. be folded into the U2 secondary Ares and Igel (1990) except for the lib ( Figure 3). :the stem-loop Ila and lib region is Nucleotides +40 to +105 of the , +40 to +100 of potato U2-22 130 of the potato U2-15 variant are ~d regions lettered A and A', and C )ase-paired sequences forming the Ila and lib (Figure 3).…”

“…:the stem-loop Ila and lib region is Nucleotides +40 to +105 of the , +40 to +100 of potato U2-22 130 of the potato U2-15 variant are ~d regions lettered A and A', and C )ase-paired sequences forming the Ila and lib (Figure 3). The under-3' represent a potential long range base-pairing interaction between B, the loop sequence of stem-loop Ila, and B', a single-stranded region between stem-loop lib and the Sin-binding site (Ares and Igel, 1990). Although primary sequence differences exist between yeast and normal potato U2snRNAs, the potential to form the same base-pairing interactions is conserved.…”

“…Three models for the secondary structure of this region have been proposed. The models predict either a single stem-loop (Keller and Noon, 1985), a pseudo-knotted structure (Ares and Igel, 1989) or a double stem-loop (Ares and Igel, 1990;Hartshorne and Agabian, 1990). Using a yeast mini-U2snRNA and a functional test for splicing, mutations have been introduced into this region to determine which of these secondary structures are most likely to form.…”