The SR splicing factors ASF/SF2 and SC35 have antagonistic effects on intronic enhancer-dependent splicing of the beta-tropomyosin alternative exon 6A.

Exons 6A and 6B of the chicken beta-tropomyosin gene are mutually exclusive and selected in a tissue-specific manner. Exon 6A is present in non-muscle and smooth muscle cells, while exon 6B is present in skeletal muscle cells. In this study we have investigated the mechanism underlying exon 6A recognition in non-muscle cells. Previous reports have identified a pyrimidine-rich intronic enhancer sequence (S4) downstream of exon 6A as essential for exon 6A 5'-splice site recognition. We show here that preincubation of HeLa cell extracts with an excess of RNA containing this sequence specifically inhibits exon 6A recognition by the splicing machinery. Splicing inhibition by an excess of this RNA can be rescued by addition of the SR protein ASF/SF2, but not by the SR proteins SC35 or 9G8. ASF/SF2 stimulates exon 6A splicing through specific interaction with the enhancer sequence. Surprisingly, SC35 behaves as an inhibitor of exon 6A splicing, since addition to HeLa nuclear extracts of increasing amounts of the SC35 protein completely abolish the stimulatory effect of ASF/SF2 on exon 6A splicing. We conclude that exon 6A recognition in vitro depends on the ratio of the ASF/SF2 to SC35 SR proteins. Taken together our results suggest that variations in the level or activity of these proteins could contribute to the tissue-specific choice of beta-tropomyosin exon 6A. In support of this we show that SR proteins isolated from skeletal muscle tissues are less efficient for exon 6A stimulation than SR proteins isolated from HeLa cells.

Tissue-specific splicing of two mutually exclusive exons of the chicken beta-tropomyosin pre-mRNA: positive and negative regulations.

Alternative splicing of premessenger RNA (pre-mRNA) is a widespread process used in higher eucaryotes to regulate gene expression. A single primary transcript can generate multiple proteins with distinct functions in a tissue- and/or developmental-specific manner. A central question in alternative splicing concerns the selection of splice sites in different cell environments. In this review, we present our results on the alternative splicing of the chicken beta-tropomyosin gene which provides an interesting model for understanding mechanisms involved in splice site selection. The beta-tropomyosin gene contains in its central portion a pair of exons (6A and 6B) that are used mutually exclusively in a tissue and developmental stage-specific manner. Exon 6A is present in mRNA of non-muscle and smooth muscle tissues while exon 6B is only present in mRNA of skeletal muscle. Regulation of both exons is necessary to ensure specific expression of beta-tropomyosin gene in non-muscle cells. Several cis-acting elements involved in the repression of exon 6B and activation of exon 6A have been identified. In addition, we show that the tissue-specific choice of exon 6A is mediated through interaction with a specific class of splicing factors, the SR proteins. In the last part of this review we will focus on possible mechanisms needed to switch to exon 6B selection in skeletal muscle tissue. We propose that tissue-specific choice of exon 6B involves down regulation of exon 6A and activation of exon 6B. A G-rich enhancer sequence downstream of exon 6B has been defined that is needed for efficient recognition of the exon 6B 5' splice site. Moreover, we suggest that alteration of the ratio between proteins of the SR family contributes to tissue-specific splice site selection.

Conformation and structural fluctuations of a 218 nucleotides long rRNA fragment: 4-thiouridine as an intrinsic photolabelling probe.

The structure in solution of an RNA fragment (218 nucleotides long) containing part of E. coli 16S rRNA domain 2 has been studied using the intrinsic photoaffinity probe 4-thiouridine (s4U). In vitro transcription with T7 polymerase, in the presence of s4U triphosphate yielded complete RNA molecules. An affinity electrophoresis system based on Phenylmercuric substituted polyacrylamide (APM) gels allows separation of the RNA chains as a function of their s4U content. Distribution of s4U within chains follows a binomial law indicating that (i) substitution is close to random, (ii) efficiency of s4U incorporation is 0.22 times that of U. The monothiolated RNA fraction isolated from APM gel was irradiated at 366 nm under native conditions and the intramolecularly crosslinked molecules, (34%), were separated on denaturing polyacrylamide gel according to loop size. The positions of the two partners of bridges were identified by mean of reverse transcription and RNA sequencing. 17 of the 41 possible s4U positions lead to detectable bridges. These crosslinks formed efficiently at the border of bihelical regions or when structural mobility is allowed. The pattern of crosslinks is in agreement with the previously proposed secondary structure but indicates that it is much more flexible than expected.

Sites of contact of mRNA with 16S rRNA and 23S rRNA in the Escherichia coli ribosome.

The locations of close encounter between ribosomal RNA (rRNA) and messenger RNA (mRNA) were determined by photochemical cross-linking experiments that employ an artificial mRNA, 51 nucleotides long, containing 14 U residues that were randomly substituted by 1-4 4-thiouridine (s4U) residues. The mRNA was bound to 70S ribosomes or 30S subunits and then was irradiated at 366 nm to activate cross-linking between the s4U residues and rRNA. Cross-linking occurred to both 16S rRNA and 23S RNA. The rRNA was then analyzed by a series of reverse transcriptase experiments to determine the locations of cross-linking. Twelve sites in the 16S rRNA and two sites in the 23S rRNA have been detected. In the 16S rRNA, two of the sites (U1381, C1395) are in the middle part of the secondary structure close to position C1400, and the remaining sites (G413, U421, G424; A532; G693; U723; A845; G1131/C1132; G1300; G1338) are distributed between six regions that are peripheral in the secondary structure. In the 23S rRNA, one site (U1065) is located in the GTPase center close to A1067, the site of thiostrepton-resistance methylation in domain II, and the other site (U887) is located a short distance away also in domain II. The distribution of these rRNA sites in the ribosome specifies an mRNA track that is consistent with other information. In addition, some of the contact points represent new constraints for the three-dimensional folding of the rRNA.

An electrophoretic method for the purification of RNA regions involved in protein crosslinking.

Direct information about structural interactions in ribonucleoprotein complexes can be obtained from crosslinking data. The purification of specific complexes, i.e., their separation from noncrosslinked proteins, from free RNA, and from other complexes, is essential for the identification of the bound proteins and the precise localization of their attachment sites in RNA. We describe a two-dimensional denaturing gel system which achieves this purification; in the first dimension basic proteins do not enter the gel and RNA--protein complexes are slowed down compared to protein free RNA, and in the second dimension sodium dodecyl sulfate improves the separation between the different complexes on the basis of their protein content.

Crosslinking of ribosomal proteins S4, S5, S7, S8, S11, S12 and S18 to domains 1 and 2 of 16S rRNA in the Escherichia coli 30S particle.

RNA-protein crosslinks were introduced into Escherichia coli 30S ribosomal subunits by treatment with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC). Complexes of 16S rRNA cross-linked to 30S ribosomal proteins were isolated and hybridized with a series of single-stranded bacteriophage M13-rDNA probes. These probes, each carrying an inserted rDNA fragment, were used to select contiguous 16S rRNA sections covering all of domain 1 and the major part of domain 2 (starting at the 5'-P terminus and ending at nucleotide 869) and the proteins covalently linked to each of these sections were identified by 2-dimensional polyacrylamide gel electrophoresis. This procedure identified proteins S4, S5, S7, S8, S11, S12, and S18 as the species most efficiently crosslinked to domains 1 and 2 of 16S rRNA. These results are discussed in the light of current knowledge of the tertiary structure of 16S rRNA in the E. coli 30S ribosomal subunit.

New RNA-protein crosslinks in domains 1 and 2 of E. coli 30S ribosomal subunits obtained by means of an intrinsic photoaffinity probe.

Functionally active 70S ribosomes containing 4-thiouridine (s4U) in place of uridine were prepared by a formerly described in vivo substitution method. Proteins were crosslinked to RNA by 366 nm photoactivation of s4U. We observe the systematic and characteristic formation of 30S dimers; they were eliminated for analysis of RNA-protein crosslinks. M13 probes containing rDNA inserts complementary to domains 1 and 2 of 16S RNA from the 5'end up to nucleotide 868 were used to select contiguous or overlapping RNA sections. The proteins covalently crosslinked to each RNA section were identified as S3, S4, S5, S7, S9, S18, S20 and S21. Several crosslinks are compatible with previously published sites for proteins S5, S18, S20 and S21; others for proteins S3, S4, S7, S9, S18 correspond necessarily to new sites.

Binding of Escherichia coli ribosomal protein S8 to 16 S rRNA. A model for the interaction and the tertiary structure of the RNA binding site.

We have investigated in detail the secondary and tertiary structures of the 16 S rRNA binding site of protein S8 using a variety of chemical and enzymatic probes. Bases were probed with dimethylsulfate (at A(N-1), C(N-3) and G(N-7)), with N-cyclohexyl-N'-(2-(N-methylmorpholino)-ethyl)-carbodiimide-p- toluenesulfonate (at G(N-1) and U(N-3)) and with diethylpyrocarbonate (at A(N-7)). The involvement of phosphates in hydrogen bonds or ion co-ordination was monitored with ethylnitrosourea. RNases T1, U2 and nuclease S1 were used to probe unpaired nucleotides and RNase V1 to monitor base-paired or stacked nucleotides. The RNA region, encompassing nucleotides 582 to 656 was probed within: (1) the complete 16 S rRNA molecule; (2) a 16 S rRNA fragment corresponding to nucleotides 578 to 756 obtained by transcription in vitro; (3) the S8-16 S rRNA complex; (4) the S8-RNA fragment complex; (5) the 30 S subunit. Cleavage or modification sites were detected by primer extension with reverse transcriptase. We present a three-dimensional model derived from mapping experiments and graphic modeling. Nucleotides in area 594-599/639-645 display unusual features: a non-canonical base-pair is formed between U598 and U641; and A595, A640 and A642 are bulging out of the major groove. The resulting helix is slightly unwound. Comparative analysis of probing experiments leads to several conclusions. (1) The synthesized fragment adopts the same conformation as the corresponding region in the complete RNA molecule, thus confirming the existence of independent folding domains in RNAs. (2) A long-range interaction involving cytosine 618 and its 5' phosphate occurs in 16 S rRNA but not in the fragment. (3) The fragment contains the complete information required for S8 binding. (4) The RNA binding site of S8 is centered in the major groove of the slightly unwound helix (594-599/639-645), with the three bulged adenines appearing as specific recognition sites. (5) This same region of the 16 S RNA is not exposed at the surface of the 30 S subunit.

Substitution of uridine in vivo by the intrinsic photoactivable probe 4-thiouridine in Escherichia coli RNA. Its use for E. coli ribosome structural analysis.

In vivo incorporation of the uridine-photoactivable analogue, 4-thiouridine, into the ribosomal RNA of an Escherichia coli pyrD strain has been demonstrated. It is highly dependent on the exogenous uridine and 4-thiouridine concentrations as well as on temperature. We have defined conditions allowing the substitution of 13 +/- 2% of the uridine residues in bulk RNA by 4-thiouridine. On a high-Mg2+ sucrose gradient, 33 +/- 3% of ribonucleic particles sediment as 70S ribosomes, the remaining being in the form of non-associated 50S and 30S particles containing immature rRNA. The thiolated 70S ribosomes tolerate a 4-5% substitution level (40 thiouridine molecules/particle). Surprisingly, 3-4% of ribosomal proteins, about two protein molecules/particle, were spontaneously covalently bound to 4-thiouridine-substituted rRNA. Specific 366-nm photoactivation increased this proportion to 10-12%, i.e. up to six or seven ribosomal protein molecules/particle. The photochemical cross-linking proceeds with apparent first-order kinetics with a quantum yield close to 5 X 10(-3). Although extensive photodynamic breakage of rRNA occurs under aerobic conditions, both the kinetics and yield of ribosomal protein cross-linking were independent of oxygenation conditions. The thiolated (4.5%) 70S ribosomes allowed the poly(U)-directed poly(Phe)synthesis at 48% the control rate. Photoactivation decreased this activity to 28% and 10% when performed under nitrogen and in aerated conditions, respectively.

Multiple crosslinks of proteins S7 and S9 to domains 3 and 4 of 16S ribosomal RNA in the Escherichia coli 30S particle.

RNA-protein cross-links were introduced into Escherichia coli 30S subunits by treatment with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide. 16S rRNA, cross-linked to 30S ribosomal proteins, was isolated and hybridized with seven single-stranded bacteriophage M13-DNA probes. These probes, each carrying an inserted rDNA fragment, were used to select contiguous RNA sections covering domains 3 and 4 (starting at nucleotide 868 and ending at the 3'OH terminus) of the 16S rRNA. The proteins covalently linked to each selected RNA section were identified by two-dimensional polyacrylamide gel electrophoresis. Proteins S7 and S9 were shown to be efficiently cross-linked to multiple sites belonging to both domains.

Multiple crosslinks of proteins S7, S9, S13 to domains 3 and 4 of 16S RNA in the 30S particle.

Functionally active 70S ribosomes containing 4-thiouracil in place of uracil (substitution level 2%) were prepared by an in vivo substitution method. RNA-protein crosslinks were introduced by 366 nm photoactivation of 4-thiouracil in the purified 30S subunits. Seven single stranded M13 probes containing rDNA inserts complementary to domains 3 and 4 of 16S RNA were constructed. These inserts approximately 100 nucleotides long starting at nucleotide 868 and ending at the 3' OH terminus were used to select contiguous RNA sections. The proteins covalently linked to each selected RNA section were identified by 2D gel electrophoresis. Proteins S7, S9, S13 were shown to be efficiently crosslinked to multiple sites belonging to both domains.

Structure of the Escherichia coli 16 S ribosomal RNA. Psoralen crosslinks and N-acetyl-N'-(p-glyoxylylbenzoyl)cystamine crosslinks detected by electron microscopy.

Escherichia coli 16 S ribosomal RNA in reconstitution buffer has been photochemically crosslinked with aminomethyltrimethylpsoralen and chemically crosslinked with N-acetyl-N'-(p-glyoxylylbenzoyl)cystamine. The positions of crosslinking have been detected by viewing the molecules in the electron microscope. DNA restriction fragments that contain psoralen mono-adducts were hybridized and crosslinked to the samples so that the orientations of the crosslinked molecules were seen directly. A two-dimensional histogram method has been used to classify the different types of looped crosslinked molecules. These methods allow the identification of 13 distinct types of loops in the photochemically crosslinked molecules and 31 distinct types of loops in the chemically crosslinked molecules. The psoralen experiments are a reinvestigation of some of our earlier results. Some of the crosslinks were previously reported in the incorrect orientation; with the corrected orientation, seven of the psoralen crosslinks can now be correlated with complementarities in the proposed secondary-structure models. However, there are still six other psoralen crosslinks that indicate additional contacts not found in the current models. The chemical crosslinks indicate pairs of single-stranded regions that must be close in the folded molecule. Many of these crosslinks occur between regions that are distant in the secondary structure; these crosslinks indicate part of the three-dimensional form of the folded molecule.

Three-dimensional arrangement of the Escherichia coli 16 S ribosomal RNA.

A model for the arrangement of the Escherichia coli 16 S ribosomal RNA in the 30 S ribosomal subunit is given. This model is based on the 16 S ribosomal RNA secondary structure, intramolecular RNA crosslinking results, protein-RNA interactions, and the locations of proteins within the 30 S subunit. These considerations allow placement of most of the RNA helices in approximate positions. The overall shape (that of an asymmetric Y) is very reminiscent of the description of the shape of the RNA made by direct determinations and is reasonably correlated to the appearance of the 30 S subunit. The identities of the three major secondary-structure domains of the 16 S ribosomal RNA are, for the most part, preserved. In addition, many close contacts between the 5' and middle RNA domains occur in the body of the particle. The 3'-terminal domain is situated in the central part of the model. This position corresponds to the region between the head and the platform structure in the 30 S subunit. The regions that represent the general locations of the messenger RNA and transfer RNA binding sites can be identified in the model.

Precise localization of several covalent RNA-RNA cross-link in Escherichia coli 16S RNA.

The RNA-RNA cross-linking reagent N-acetyl-N'-(p-glyoxylyl-benzoyl)cystamine, which reacts via its glyoxal residue with guanines not involved in G X C base pairs, has been used to introduce reversible RNA-RNA cross-links into Escherichia coli 16S rRNA. A two-dimensional gel method has been devised for the separation of the cross-linked oligonucleotides, and the precise location of guanines involved in four of these cross-links has been determined by sequencing the oligonucleotides. One cross-link involves guanosines 1315 and 1360 situated in two hairpin end loops of domain III. The other cross-links involves pairs of guanosine situated within the same hairpin end loops.

Protein-RNA crosslinking in Escherichia coli 30S ribosomal subunits. Identification of a 16S rRNA fragment crosslinked to protein S12 by the use of the chemical crosslinking reagent 1-ethyl-3-dimethyl-aminopropylcarbodiimide.

1-ethyl-3-dimethyl aminopropylcarbodiimide (EDC) was used to cross-link 30S ribosomal proteins to 16S rRNA within the E. coli 3OS ribosomal subunit. Covalently linked complexes containing 30S proteins and 16S rRNA, isolated by sedimentation of dissociated crosslinked 30S subunits through SDS containing sucrose gradients, were digested with RNase T1, and the resulting oligonucleotide-protein complexes were fractionated on SDS containing polyacrylamide gels. Eluted complexes containing 30S proteins S9 and S12 linked to oligonucleotides were obtained in pure form. Oligonucleotide 5'terminal labelling was successful in the case of S12 containing but not of the S9 containing complex and led to identification of the S12 bound oligonucleotide as CAACUCG which is located at positions 1316-1322 in the 16S rRNA sequence. Protein S12 is crosslinked to the terminal G of this heptanucleotide.

Properties of ribosomes and ribosomal RNAs synthesized by Escherichia coli grown in the presence of ethionine. Normal maturation of ribosomal RNA in the absence of methylation.

Analysis of 16-S rRNA synthesized in Escherichia coli D10 (met-) incubated in a medium containing ethionine in place of methionine shows that it lacks most and probably all of the methyl groups present in normal 16-SrRNA but possesses the same 3'-OH, and 5'-phosphate terminal sequences as the latter. 23-S rRNA formed in ethionine-treated cells also contains normal terminal sequences. 5-S rRNAs of normal and ethionine-treated E. coli D10 are identical. These results lead to the conclusion that methylation of ribosomal precursor RNAs is not necessary for their maturation to products with normal chain lengths and does not influence the conformation of 16-S rRNA.