The 3-5 riboexonuclease Rrp6p, a nuclear component of the exosome, functions with other exosome components to produce the mature 3 ends of 5.8S rRNA, sno-and snRNAs, and to destroy improperly processed precursor (pre)-rRNAs and pre-mRNAs. Rrp6p is a member of the RNase D family of riboexonucleases and displays a high degree of homology with the active site of the deoxyriboexonuclease domain of Escherichia coli DNA polymerase I, the crystal structure of which indicates a two-metal ion mechanism for phosphodiester bond hydrolysis. Mutation of each of the conserved residues predicted to coordinate metal ions in the active site of Rrp6p abolished activity of the enzyme in vitro and in vivo. Complete loss of Rrp6p activity caused by the Y361F and Y361A mutations supports the critical role proposed for the phenolic hydroxyl of Tyr361 in the reaction mechanism. Rrp6p also contains an helicase RNase D C-terminal (HRDC) domain of unknown function that is similar to domains in the Werner's and Bloom's Syndrome proteins. A point mutation in this domain results in Rrp6p that localizes to the nucleus, but fails to efficiently process the 3 ends of 5.8S pre-rRNA and some pre-snoRNAs. In contrast, this mutant retains the ability to degrade rRNA processing intermediates and 3-extended, poly(A)+ snoRNAs. These findings indicate the potential for independent control of the processing and degradation functions of Rrp6p.

Batten disease, an inherited neurodegenerative storage disease affecting children, results from the autosomal recessive inheritance of mutations in Cln3. The function of the CLN3 protein remains unknown. A key to understanding the pathology of this devastating disease will be to elucidate the function of CLN3 at the cellular level. CLN3 has proven difficult to study as it is predicted to be a membrane protein expressed at relatively low levels. This article is a critical review of various approaches used in examining the structure, trafficking, and localization of CLN3. We conclude that CLN3 is likely resident in the lysosomal/endosomal membrane. Different groups have postulated conflicting orientations for CLN3 within this membrane. In addition, CLN3 undergoes several posttranslational modifications and is trafficked through the endoplasmic reticulum and Golgi. Recent evidence also suggests that CLN3 traffics via the plasma membrane. Although the function of this protein remains elusive, it is apparent that genetic alterations in Cln3 may have a direct affect on lysosomal function.

Exoribonucleases function in the processing and degradation of a variety of RNAs in all organisms. These enzymes play a particularly important role in the maturation of rRNAs and in a quality-control pathway that degrades rRNA precursors upon inhibition of ribosome biogenesis. Strains with defects in 3 0 -5 0 exoribonucleolytic components of the RNA processing exosome accumulate polyadenylated precursor rRNAs that also arise in strains with ribosome biogenesis defects. These findings suggested that polyadenylation might target pre-rRNAs for degradation by the exosome. Here we report experiments that indicate a role for the 5 0 -3 0 exoribonuclease Rat1p and its associated protein Rai1p in the degradation of poly(A) + pre-rRNAs. Depletion of Rat1p enhances the amount of poly(A) + pre-rRNA that accumulates in strains deleted for the exosome subunit Rrp6p and decreases their 5 0 heterogeneity. Deletion of RAI1 results in the accumulation of poly(A) + pre-rRNAs, and inhibits Rat1p-dependent 5 0 -end processing and Rrp6p-dependent 3 0 -end processing of 5.8S rRNA. RAT1 and RAI1 mutations cause synergistic growth defects in the presence of rrp6-D, consistent with the interdependence of 5 0 -end and 3 0 -end processing pathways. These findings suggest that Rai1p may coordinate the 5 0 -end and 3 0 -end processing and degradation activities of Rat1p and the nuclear exosome.

The neuronal ceroid lipofuscinoses (NCLs) are neurodegenerative disorders. Nevertheless, small model organisms, including those lacking a nervous system, have proven invaluable in the study of mechanisms that underlie the disease and in studying the functions of the conserved proteins associated to each disease. From the single-celled yeast, Saccharomyces cerevisiae and Schizosaccharomyces pombe, to the worm, Caenorhabditis elegans and the fruitfly, Drosophila melanogaster, biochemical and, in particular, genetic studies on these organisms have provided insight into the NCLs.