Gene, 148 (1994) 59966 0 1994 Elsevier Science B.V. All rights reserved. 0378-l 119/94/$07.00 59 zyxwvutsr GENE 08180 Analysis of the yeast NSRl gene and protein domain comparison between Nsr 1 and human hnRNP type Al (RNA-binding proteins; RNA recognition motif; zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Saccharomy ces cerevisiae; nucleolar protein; pre-rRNA processing; gene expression, yeast transcriptional unit) Chiara Gamberi, Giovanna Contreas, Maria Grazia Romanelli and Carlo Morandi lstituto di Scienze Biologiche, Facoltd di Medicina e Chirurgia. Universitirdi Verona. l-37134 Verona, Italy. Received by G. Bernardi: 11 February 1994; Revised/Accepted: 30 May/2 June 1994; Received at publishers: 13 June 1994 SUMMARY The yeast nucleolar protein-encoding gene NSRl was isolated by low-stringency screening of a yeast genomic library with the human heterogeneous nuclear ribonucleoprotein type Al (hnRNP Al) cDNA probe, and was mapped to chromosome VII. RNA abundance was determined and the transcription start point and polyadenylation site were mapped. A comparison between the Nsrl and hnRNP Al proteins, based on homopolymer RNA binding to their structural domains in vitro, revealed a striking biochemical similarity. When the N-terminal, lysine- and arginine-rich domain of Nsrl was removed, the truncated protein behaved similarly to hnRNP Al; furthermore, the two RRM (RNA recognition motif) domains of Nsrl behaved in the same manner as the two RRM domains of hnRNP Al. The biochemical data, therefore, would support the hypothesis that the two RRM domains in hnRNP Al and Nsrl interact with RNA in a similar manner in both mammalian and yeast cells, respectively. INTRODUCTION The yeast nuclear signal recognition protein Nsrl is a 44-kDa nucleolar protein containing two RRM domains. It has been demonstrated by gene disruption, that Nsrl is directly involved in ribosome biogenesis, at the level of rRNA maturation (Lee et al., 1991). More recently, Nsrl has been shown to be a cold-shock induced protein (Kondo et al., 1992), and thought to be similar in function to mammalian nucleolin (Lapeyre et al., 1987). Nsrl Correspondence to: Dr. C. Morandi, Istituto di Scienze Biologiche, Facolta di Medicina e Chirurgia, Universita di Verona, Strada Le Grazie, I-37134 Verona, Italy. Tel. (39-45) 809-8186; Fax (39-45) 809-8180; e-mail: morand@ivruniv.bitnet Abbreviations: aa, amino acid(s); AP, adapter primer; bp, base pair(s); CHEF, contour-clamped homogeneous electric field; cpm, counts per minute; A, deletion; E., Escherichia; CAR, glycine- and arginine-rich; Carl, yeast GAR protein 1; GSP, gene-specific primer; hnRNP, hetero- geneous nuclear RNP; HRBA, homopolymer RNA-binding assay; kb, kilobase or 1000 bp; NGSP, nested GSP; Nopl, yeast nucleolar pro- SSDI 037X-l 119(94)00391-5 defective mutants show a phenotype essentially identical to that of strains depleted of Nopl and Garl proteins and of the snoRNAs U3 and U14; in fact they have an abnormal processing of 3% pre-rRNA ultimately leading to a reduction of the 18s rRNA (Kondo and Inouye; 1992, Lee et al., 1992). HnRNP type Al core protein is a 34-kDa nuclear poly- peptide that associates with the newly transcribed hnRNA in the 40s post-transcriptional complex and shuttles between the nucleoplasm and the cytoplasm in tein 1; Nsrl, nuclear signal recognition protein 1: NSRI, gene encoding Nsrl; nt, nucleotide(s); OFAGE, orthogonal-field alternate gel electro- phoresis; oligo, oligodeoxyribonucleotide; ORF, open reading frame: PAGE, polyacrylamide-gel electrophoresis; PCR. polymerase chain reaction: RACE, rapid amplification of cDNA ends; RGG, Arg-Gly- Gly; RNA-BP, RNA-binding protein; RNP, ribonucleoprotein; RRM, RNA recognition motif; S, sedimentation constant; S., Saccharomy crs; SDS, sodium dodecyl sulfate; snoRNA, small nucleolar RNA; snRNP, small nuclear RNP; Ssbl, S. cereuisiae single-stranded DNA-binding protein 1; tsp, transcription start point(s); UP I, proteolytrc fragment of the hnRNP Al protein.