Nucleolar localization of the yeast RNA exosome subunit Rrp44 hints at early pre-rRNA processing as its main function.

The RNA exosome is a multisubunit protein complex involved in RNA surveillance of all classes of RNA, and is essential for pre-rRNA processing. The exosome is conserved throughout evolution, present in archaea and eukaryotes from yeast to humans, where it localizes to the nucleus and cytoplasm. The catalytically active subunit Rrp44/Dis3 of the exosome in budding yeast (Saccharomyces cerevisiae) is considered a protein present in these two subcellular compartments, and here we report that it not only localizes mainly to the nucleus, but is concentrated in the nucleolus, where the early pre-rRNA processing reactions take place. Moreover, we show by confocal microscopy analysis that the core exosome subunits Rrp41 and Rrp43 also localize largely to the nucleus and strongly accumulate in the nucleolus. These results shown here shed additional light on the localization of the yeast exosome and have implications regarding the main function of this RNase complex, which seems to be primarily in early pre-rRNA processing and surveillance.

The ribosome assembly factor Nop53 controls association of the RNA exosome with pre-60S particles in yeast.

Eukaryotic ribosomal biogenesis is a high-energy-demanding and complex process that requires hundreds of trans-acting factors to dynamically build the highly-organized 40S and 60S subunits. Each ribonucleoprotein complex comprises specific rRNAs and ribosomal proteins that are organized into functional domains. The RNA exosome complex plays a crucial role as one of the pre-60S-processing factors, because it is the RNase responsible for processing the 7S pre-rRNA to the mature 5.8S rRNA. The yeast pre-60S assembly factor Nop53 has previously been shown to associate with the nucleoplasmic pre-60S in a region containing the "foot" structure assembled around the 3' end of the 7S pre-rRNA. Nop53 interacts with 25S rRNA and with several 60S assembly factors, including the RNA exosome, specifically, with its catalytic subunit Rrp6 and with the exosome-associated RNA helicase Mtr4. Nop53 is therefore considered the adaptor responsible for recruiting the exosome complex for 7S processing. Here, using proteomics-based approaches in budding yeast to analyze the effects of Nop53 on the exosome interactome, we found that the exosome binds pre-ribosomal complexes early during the ribosome maturation pathway. We also identified interactions through which Nop53 modulates exosome activity in the context of 60S maturation and provide evidence that in addition to recruiting the exosome, Nop53 may also be important for positioning the exosome during 7S processing. On the basis of these findings, we propose that the exosome is recruited much earlier during ribosome assembly than previously thought, suggesting the existence of additional interactions that remain to be described.

Identification of karyopherins involved in the nuclear import of RNA exosome subunit Rrp6 in Saccharomyces cerevisiae.

The exosome is a conserved multiprotein complex essential for RNA processing and degradation. The nuclear exosome is a key factor for pre-rRNA processing through the activity of its catalytic subunits, Rrp6 and Rrp44. In Saccharomyces cerevisiae, Rrp6 is exclusively nuclear and has been shown to interact with exosome cofactors. With the aim of analyzing proteins associated with the nuclear exosome, in this work, we purified the complex with Rrp6-TAP, identified the co-purified proteins by mass spectrometry, and found karyopherins to be one of the major groups of proteins enriched in the samples. By investigating the biological importance of these protein interactions, we identified Srp1, Kap95, and Sxm1 as the most important karyopherins for Rrp6 nuclear import and the nuclear localization signals recognized by them. Based on the results shown here, we propose a model of multiple pathways for the transport of Rrp6 to the nucleus.