Search

The three-dimensional structures of chymotrypsin- and methylamine-treated negatively stained human α 2 -macroglobulin have been determined by weighted back projection from electron microscope data. Projections of the reconstructions show good concordance with two-dimensional averages of both stained and frozen-hydrated molecules. The reconstructions reveal that the H-shaped front projection of the molecule is related to the smaller ellipsoidal end view by a rotation of 90° about the crossbar (minor axis) of the H. This finding is in agreement with tilt studies. The reconstruction of the α 2 -macroglobulin-methylamine reveals an hour-glass shaped void which is filled by the two proteinase molecules in the reconstruction of α 2 -macroglobulin-chymotrypsin. Protein plugs which appear to block the exterior entrances to the cavity may function to prevent access of proteins to the encapsulated proteinase and serve to block its escape. Extensive thresholding of each reconstruction leaves a “backbone” consisting of two side-by-side rod-like structures, suggesting that this is the arrangement of the two protomeric units which form the molecule. Both structures show some departure from the expected symmetry. The asymmetries are robust features of the reconstructions and may reflect structurally asymmetric features of the transformation from the native to the chymotrypsin-treated form of the molecule.

Splice site selection is a key element of pre-mRNA splicing. Although it is known to involve specific recognition of short consensus sequences by the splicing machinery, the mechanisms by which 5′ splice sites are accurately identified remain controversial and incompletely resolved. The human F7 gene contains in its seventh intron (IVS7) a 37-bp VNTR minisatellite whose first element spans the exon7–IVS7 boundary. As a consequence, the IVS7 authentic donor splice site is followed by several cryptic splice sites identical in sequence, referred to as 5′ pseudo-sites, which normally remain silent. This region, therefore, provides a remarkable model to decipher the mechanism underlying 5′ splice site selection in mammals. We previously suggested a model for splice site selection that, in the presence of consecutive splice consensus sequences, would stimulate exclusively the selection of the most upstream 5′ splice site, rather than repressing the 3′ following pseudo-sites. In the present study, we provide experimental support to this hypothesis by using a mutational approach involving a panel of 50 mutant and wild-type F7 constructs expressed in various cell types. We demonstrate that the F7 IVS7 5′ pseudo-sites are functional, but do not compete with the authentic donor splice site. Moreover, we show that the selection of the 5′ splice site follows a scanning-type mechanism, precluding competition with other functional 5′ pseudo-sites available on immediate sequence context downstream of the activated one. In addition, 5′ pseudo-sites with an increased complementarity to U1snRNA up to 91% do not compete with the identified scanning mechanism. Altogether, these findings, which unveil a cell type–independent 5′−3′-oriented scanning process for accurate recognition of the authentic 5′ splice site, reconciliate apparently contradictory observations by establishing a hierarchy of competitiveness among the determinants involved in 5′ splice site selection., Synopsis Typically, mammalian genes contain coding sequences (exons) separated by non-coding sequences (introns). Introns are removed during pre-mRNA splicing. The accurate recognition of introns during splicing is essential, as any abnormality in that process will generate abnormal mRNAs that can cause diseases. Understanding the mechanisms of accurate splice site selection is of prime interest to life scientists. Exon–intron borders (splice sites) are defined by short sequences that are poorly conserved. The strength of any splice sequence can be assessed by its degree of homology with a splice site consensus sequence. Within exons and introns, several sequences can match with this consensus as well as or better than the splice sites. Using a system in which a splice site sequence is repeated several times in the intron, the authors showed that linear 5′−3′ search is a leading mechanism underlying splice site selection. This scanning mechanism is cell type–independent, and only the most upstream splice site of all the series is selected, even if splice sites with a better match to the consensus are in the vicinity. These findings reconciliate contradictory observations and establish a hierarchy among the determinants involved in splice site selection.