Binding of the nucleolar protein Nep1 to a specific site in ribosomal RNA and isolation of deltasnr57 and multicopy RPS19 as nep1 suppressors.
Markus Buchhaupt, Britta Meyer, Peter Kötter, Karl-Dieter Entian
Institute of Microbiology, JW-Goethe University, Marie-Curie-Str. 9, Frankfurt am Main, 60439, Germany
The nucleolar protein Nep1 was previously shown to be involved in the process of pre-rRNA processing and furthermore to infere directly or indirectly with a methylation reaction. By performing a three-hybrid screen, we were now able to isolate several short RNA sequences that show affinity to Nep1 and to define a minimal RNA structure that binds to the protein. One of the sequences identified contains the nucleotides 1553-1577 of the 18S rRNA molecule and corresponds most probably to the physiological RNA ligand. As the base moiety of G1573 in the 18S rRNA of mature ribosomes contains a posttranscriptionally introduced methyl group, Nep1 could perhaps be responsible for that modification. To investigate this possibility we want to analyze 18S rRNA out of a yeast strain in which NEP1 had been deleted. Due to the fact that NEP1 is essential, we had to isolate deltanep1 suppressor mutations, of which two could also bring interesting insights for the Nep1 function during ribosome biogenesis. So the loss of SNR57, which encodes the small nucleolar RNA, that guides 2'O-ribose methylation of G1570, can partially suppress nep1 mutants. Moreover we discovered that multicopy expression of the ribosomal protein Rps19b has a partial suppression effect, which may lead to the hypothesis, that the Nep1 function could be a prerequisite for association of Rps19 with preribosomal particles.
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