Exonic splicing regulatory sequences (ESRs) are cis-acting factor binding sites that regulate constitutive and alternative splicing. A computational method based on the conservation level of wobble positions and the overabundance of sequence motifs between 46,103 human and mouse orthologous exons was developed, identifying 285 putative ESRs. Alternatively spliced exons that are either short in length or contain weak splice sites show the highest conservation level of those ESRs, especially toward the edges of exons. ESRs that are abundant in those subgroups show a different distribution between constitutively and alternatively spliced exons. Representatives of these ESRs and two SR protein binding sites were shown, experimentally, to display variable regulatory effects on alternative splicing, depending on their relative locations in the exon. This finding signifies the delicate positional effect of ESRs on alternative splicing regulation.

Human-mouse comparative genomics is an informative tool to assess sequence functionality as inferred from its conservation level. We used this approach to examine dependency among different positions of the 5 splice site. We compiled a data set of 50,493 homologous human-mouse internal exons and analyzed the frequency of changes among different positions of homologous human-mouse 5 splice-site pairs. We found mutual relationships between positions +4 and +5, +5 and +6, −2 and +5, and −1 and +5. We also demonstrated the association between the exonic and the intronic positions of the 5 splice site, in which a stronger interaction of U1 snRNA and the intronic portion of the 5 splice site compensates for weak interaction of U1 snRNA and the exonic portion of the 5 splice site, and vice versa. By using an ex vivo system that mimics the effect of mutation in the 5 splice site leading to familial dysautonomia, we demonstrated that U1 snRNA base-pairing with positions +6 and −1 is the only functional requirement for mRNA splicing of this 5 splice site. Our findings indicate the importance of U1 snRNA base-pairing to the exonic portion of the 5 splice site.

In cytochrome c, ligation of the heme iron by the methionine-80 sulfur plays a major role in determining the structure and the thermodynamic stability of the protein. In the ferric state, this bond is reversibly broken by moderately acid or alkaline pH's (pK's 2.5 and 9.4, respectively) and by exogenous ligands. NMR studies of horse ferricytochrome c in which the Met-65 and Met-80 methyl groups were chemically enriched with 13C demonstrate that, at 59 degrees C, a temperature at which the protein is still folded, the sulfur-iron bond is already partially broken. This structural change corresponds to the reversible disappearance upon moderate heating of the 695 nm band, characteristic of the sulfur-iron coordination of this protein. The thermal effect results from a shift in the alkaline pK from 9.4 at 25 degrees C to 8.2 at 59 degrees C. The exchange rate from iron-bound to free methionine-80 at 59 degrees C is 1.8 s-1, as measured by saturation transfer experiments. The free and bound methionine-80 epsilon-methyl groups in the 1H spectrum are assigned as (1.87, 2.25) and -21.43, respectively; in the 13C spectrum they are assigned as 15.6 and 12.8, respectively (all these values are in ppm from 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, sodium salt).

The ~peetrul chanties caused by bindintt ~ort liLlnnd~ tt~ the ¢yt,~¢itrt~me ¢ iron =uld their ¢offehttit~n to lil~and allinitie~ ~uppt~rt the hypothe~i~ that the iron-m~thionlne ~ulfi~r bond of thi~ heine protein i~ enhanced by deloeali~ation or the metal t.~t ~le~tron~ into the empty 3d orhltnl~ of" the lisand aton~, The~e findins~ ~tlxo explain the unique ~peetrtim or cyt(~d~r~me ¢ in tile fiw red.Cylochromc ¢; St~ft ligands; Iron-sulfur hond

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A multicomponent complex of proteins and RNA is assembled on the newly synthesized pre-mRNA to form the spliceosome. This complex catalyzes a two-step transesterification reaction required to remove the introns and ligate the exons. To date, only six proteins have been found necessary for the second step of splicing in yeast, and their human homologs have been identified. We demonstrate that the addition of the selective chelator of zinc, 1,10-phenanthroline, to an in vitro mRNA splicing reaction causes a dose-dependent inhibition of the second step of splicing. This inhibition is accompanied by the accumulation of spliceosomes paused before completion of step two of the splicing reaction. The inhibition effect on the second step is due neither to snRNA degradation nor to direct binding to the mRNA, and is reversible by dialysis or add-back of zinc, but not of other divalent metals, at the beginning of the reaction. These findings suggest that the activity of a putative zinc-dependent metalloprotein(s) involved in the second step of splicing is affected. This study outlines a new method for specific reversible inhibition of the second step of splicing using external reagents, and suggests a possible role of divalent cations in the second step of mRNA splicing, most likely zinc.

Asn-52 of rat cytochrome c and baker's yeast iso-1-cytochrome c was changed to isoleucine by site-directed mutagenesis and the mutated proteins expressed in and purified from cultures of transformed yeast. This mutation affected the affinity of the haem iron for the Met-80 sulphur in the ferric state and the reduction potential of the molecule. The yeast protein, in which the sulphur-iron bond is distinctly weaker than in vertebrate cytochromes c, became very similar to the latter: the pKa of the alkaline ionization rose from 8.3 to 9.4 and that of the acidic ionization decreased from 3.4 to 2.8. The rates of binding and dissociation of cyanide became markedly lower, and the affinity was lowered by half an order of magnitude. In the ferrous state the dissociation of cyanide from the variant yeast cytochrome c was three times slower than in the wild-type. The same mutation had analogous but less pronounced effects on rat cytochrome c: it did not alter the alkaline ionization pKa nor its affinity for cyanide, but it lowered its acidic ionization pKa from 2.8 to 2.2. These results indicate that the mutation of Asn-52 to isoleucine increases the stability of the cytochrome c closed-haem crevice as observed earlier for the mutation of Tyr-67 to phenylalanine [Luntz, Schejter, Garber and Margoliash (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3524-3528], because of either its effects on the hydrogen-bonding of an interior water molecule or a general increase in the hydrophobicity of the protein in the domain occupied by the mutated residues. The reduction potentials were affected in different ways; the Eo of rat cytochrome c rose by 14 mV whereas that of the yeast iso-1 cychrome c was 30 mV lower as a result of the change of Asn-52 to isoleucine.

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