The rodent alphaA-crystallin gene: mutagenesis of a non-consensus 5'-splice site to study alternative splicing in vivo.

alphaA-Crystallin is a member of the small heat shock protein family that is abundantly expressed as a structural component in the vertebrate eye lens. In lenses of rodents and some other mammals, there occurs a minor variant of alphaA-crystallin, which has an insertion of 23 amino acid residues. This variant, alphaA(ins)-crystallin, results from differential integration of an optional exon into a small fraction of the mRNA. We have studied whether this alternative splicing is caused by a non-consensus cytosine in the 5' splice site adjacent to the optional exon. After replacement of the aberrant cytosine in the hamster alphaA-crystallin gene by a consensus thymine, and transient transfection of this gene in Chinese Hamster Ovary cells, the optional exon is indeed almost completely spliced into the mature mRNA. In contrast, replacement of the cytosine by adenine or guanine completely abolishes the splicing of the optional exon. Our results confirm that alternative splicing of the alphaA-crystallin primary transcript is mainly due to a non-consensus 5' splice site nucleotide. However, we conclude that the small size of the optional exon is probably an additional contributing factor and therefore it seems that the splicing mechanism is based on recognition of exons rather than introns.

alpha B-crystallin and hsp25 in neonatal cardiac cells--differences in cellular localization under stress conditions.

Two members of the small heat shock protein family, alpha B-crystallin and hsp25, occur at high levels in the mammalian heart. To try and understand any differences in functioning, we compared their properties in cultured rat neonatal cardiac myocytes. Both proteins are stress-inducible, but the level of hsp25 is only slightly increased in cultured cardiac myocytes subjected to hyperthermic stress, while alpha B-crystallin levels even remain unchanged. Phosphorylation of alpha B-crystallin and to a lesser extent also of hsp25 is induced after the heat shock. Directly after heat stress, alpha B-crystallin and hsp25 are partly found in detergent-insoluble fractions, representing cytoskeletal/nuclear structures. Additionally, we show by confocal laser scanning microscopy that alpha B-crystallin and hsp25 become associated with sarcomeric structures directly after the heat shock, indicating a cytoskeletal protective function. Four to six hours after the heat shock, both proteins reoccupy their original positions in the cytoplasm again. In contrast to alpha B-crystallin, hsp25 not only translocates to the cytoskeleton but also migrates to positions inside the nucleus. Despite the fact that both proteins are normally part of the same complex, their behavior in neonatal cardiac myocytes appears to be very different. The sarcomeric association of alpha B-crystallin occurs under milder conditions and persists for a longer period of time in comparison with hsp25. Our findings suggest that alpha B-crystallin and hsp25 are both involved in protection of the cytoskeleton during stress situations in the heart, although in different manners. In addition, hsp25 also plays a role inside the nucleus.

The mammalian small heat-shock protein Hsp20 forms dimers and is a poor chaperone.

Hsp20 is one of the newly described members of the mammalian small heat-shock protein (sHsp) family. It occurs most abundantly in skeletal muscle and heart. We isolated clones for Hsp20 from a rat heart cDNA library, and expressed the protein in Escherichia coli to characterize this little known sHsp. Recombinant Hsp20 displayed similar far-ultraviolet circular dichroism spectra as the most closely related sHsp, alpha B-crystallin, but was less heat stable, denaturing upon heating to 50 degrees C. While other mammalian recombinant sHsps form large multimeric complexes, Hsp20 occurs in two complex sizes, 43-kDa dimers and 470-kDa multimers. The ratio between the two forms depends on protein concentration. Moreover, Hsp20 has a much lower chaperone-like activity than alpha B-crystallin, as indicated by its relatively poor capacity to diminish the reduction-induced aggregation of insulin B chains. Hsp20 is considerably shorter at the C-terminus and less polar than other sHsps, but 1H-NMR spectroscopy reveals that the last 10 residues are flexible, as in the other sHsps. Our findings suggest that Hsp20 is a special member of the sHsp family in being less heat stable and tending to form dimers. These properties, together with the shorter and less polar C-terminal extension, may contribute to the less effective chaperone-like activity.