USH3A transcripts encode clarin-1, a four-transmembrane-domain protein with a possible role in sensory synapses.

Usher syndrome type 3 (USH3) is an autosomal recessive disorder characterised by the association of post-lingual progressive hearing loss, progressive visual loss due to retinitis pigmentosa and variable presence of vestibular dysfunction. Because the previously defined transcripts do not account for all USH3 cases, we performed further analysis and revealed the presence of additional exons embedded in longer human and mouse USH3A transcripts and three novel USH3A mutations. Expression of Ush3a transcripts was localised by whole mount in situ hybridisation to cochlear hair cells and spiral ganglion cells. The full length USH3A transcript encodes clarin-1, a four-transmembrane-domain protein, which defines a novel vertebrate-specific family of three paralogues. Limited sequence homology to stargazin, a cerebellar synapse four-transmembrane-domain protein, suggests a role for clarin-1 in hair cell and photoreceptor cell synapses, as well as a common pathophysiological pathway for different Usher syndromes.

Distribution of split DnaE inteins in cyanobacteria.

Inteins are genetic elements found inside the coding regions of different host proteins and are translated in frame with them. The intein-encoded protein region is removed by an autocatalytic protein-splicing reaction that ligates the host protein flanks with a peptide bond. This reaction can also occur in trans with the intein and host protein split in two. After translation of the two genes, the two intein parts ligate their flanking protein parts to each other, producing the mature protein. Naturally split inteins are only known in the DNA polymerase III alpha subunit (polC or dnaE gene) of a few cyanobacteria. Analysing the phylogenetic distribution and probable genetic propagation mode of these split inteins, we conclude that they are genetically fixed in several large cyanobacterial lineages. To test our hypothesis, we sequenced parts of the dnaE genes from five diverse cyanobacteria and found all species to have the same type of split intein. Our results suggest the occurrence of a genetic rearrangement in the ancestor of a large division of cyanobacteria. This event fixed the dnaE gene in a unique two-genes one-protein configuration in the progenitor of many cyanobacteria. Our hypothesis, findings and the cloning procedure that we established allow the identification and acquisition of many naturally split inteins. Having a large and diverse repertoire of these unique inteins will enable studies of their distinct activity and enhance their use in biotechnology.

Distribution and function of new bacterial intein-like protein domains.

Hint protein domains appear in inteins and in the C-terminal region of Hedgehog and Hedgehog-like animal developmental proteins. Intein Hint domains are responsible and sufficient for protein-splicing of their host-protein flanks. In Hedgehog proteins the Hint domain autocatalyses its cleavage from the N-terminal domain of the Hedgehog protein by attaching a cholesterol molecule to it. We identified two new types of Hint domains. Both types have active site sequence features of Hint domains but also possess distinguishing sequence features. The new domains appear in more than 50 different proteins from diverse bacteria, including pathogenic species of humans and plants, such as Neisseria meningitidis and Pseudomonas syringae. These new domains are termed bacterial intein-like (BIL) domains. Bacterial intein-like domains are present in variable protein regions and are typically flanked by domains that also appear in secreted proteins such as filamentous haemagglutinin and calcium binding RTX repeats. Phylogenetic and genomic analysis of BIL sequences suggests that they were positively selected for in different lineages. We cloned two BIL domains of different types and showed them to be active. One of the domains efficiently cleaved itself from its C-terminal flank and could also protein-splice its two flanks, in E. coli and in a cell free system. We discuss several possible biological roles for BIL domains including microevolution and post translational modification for generating protein variability.