Abstract. We previously identified a protein (p67) in the yeast, Saccharomyces cerevisiae, that specifically recognizes nuclear localization sequences. We report here the partial purification of p67, and the isolation, sequencing, and disruption of the gene (NSR1) encoding this protein, p67 was purified using an affinity column conjugated with a peptide containing the histone H2B nuclear localization sequence from yeast. Using antibodies against p67 we have cloned the gene for this protein. The protein encoded by the NSR1 gene recognizes the wild-type H2B nuclear localization sequence, but does not recognize a mutant H2B sequence that is incompetent for nuclear localization in vivo. Interestingly, the NSRI protein has two RNA recognition motifs, as well as an acidic NH2 terminus containing a series of serine clusters, and a basic COOH terminus containing arg-gly repeats. We have confirmed the nuclear localization of p67 by immunofluorescence and found that a restricted portion of the nucleus is highlighted. We have also shown that NSRI (p67) is required for normal cell growth.

NSR1 is a yeast nuclear localization sequence-binding protein showing striking similarity in its domain structure to nucleolin. Cells lacking NSR1 are viable but have a severe growth defect. We show here that NSR1, like nucleolin, is involved in ribosome biogenesis. The nsrl mutant is deficient in pre-rRNA processing such that the initial 35S pre-rRNA processing is blocked and 20S pre-rRNA is nearly absent. The reduced amount of 20S pre-rRNA leads to a shortage of 18S rRNA and is reflected in a change in the distribution of 60S and 40S ribosomal subunits; there is no free pool of 40S subunits, and the free pool of 60S subunits is greatly increased in size. The lack of free 40S subunits or the improper assembly of these subunits causes the nsrl mutant to show sensitivity to the antibiotic paromomycin, which affects protein translation, at concentrations that do not affect the growth of the wild-type strain. Our data support the idea that NSR1 is involved in the proper assembly of pre-rRNA particles, possibly by bringing rRNA and ribosomal proteins together by virtue of its nuclear localization sequence-binding domain and multiple RNA recognition motifs. Alternatively, NSR1 may also act to regulate the nuclear entry of ribosomal proteins required for proper assembly of pre-rRNA particles.In eukaryotic cells, the nucleolus is a specialized subcompartment in which ribosome biogenesis occurs. Pre-rRNA first is transcribed from rDNA genes located in the nucleolus and then undergoes a series of modifications. Ribosomal proteins are imported from the cytoplasm and packed onto the RNA molecule. At the same time, the primary transcript goes through sequential cleavages to generate mature forms of rRNA. Finally, the newly formed ribosomal particles are exported to the cytoplasm.In the yeast Saccharomyces cerevisiae, the largest detectable transcript from the rDNA genes is a 35S pre-rRNA (17), which is rapidly processed into three molecules: the 18S rRNA assembled into 40S ribosomal subunits and the 5.8S and 25S rRNAs found in 60S subunits (Fig. 1). During or immediately after transcription of the rDNA genes, the pre-rRNA is modified, mainly by methylation (32, 34). Meanwhile, a large number of ribosomal proteins and nonribosomal components (including proteins and RNA) associate with the pre-rRNA to form a 90S preribosomal particle. This particle is then split into 66S and 43S preribosomal particles, which are the precursors of the 60S and 40S subunits, respectively (31). While the 66S particles mature completely within the nucleus, the final maturation steps of the 43S particles, including the processing of 20S to 18S rRNA, are completed in the cytoplasm (31,33).Only a few mutations that block specific steps in the pre-rRNA processing pathway have been found. One example is a temperature-sensitive mutation in a gene designated RRPJ that was shown to prevent the 27S-to-25S rRNA cleavage step specifically (1). In another mutant, CLP-8, the efficiency of the processing of 20S to 18S rRNA is greatly reduced, apparently because...

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The RASopathies are a group of genetic syndromes caused by germline mutations in genes that encode components of the Ras/mitogen-activated protein kinase (MAPK) pathway. Some of these syndromes are neurofibromatosis type 1, Noonan syndrome, Costello syndrome, cardio-facio-cutaneous syndrome, LEOPARD syndrome and Legius syndrome. Their common underlying pathogenetic mechanism brings about significant overlap in phenotypic features and includes craniofacial dysmorphology, cardiac, cutaneous, musculoskeletal, GI and ocular abnormalities, and a predisposition to cancer. The proceedings from the symposium “Genetic Syndromes of the Ras/MAPK Pathway: From Bedside to Bench and Back” chronicle the timely and typical research symposium which brought together clinicians, basic scientists, physician-scientists, advocate leaders, trainees, students and individuals with Ras syndromes and their families. The goals, to discuss basic science and clinical issues, to set forth a solid framework for future research, to direct translational applications towards therapy and to set forth best practices for individuals with RASopathies was successfully meet with a commitment to begin to move towards clinical trials.