Structural requirements of 5S rRNA for nuclear transport, 7S ribonucleoprotein particle assembly, and 60S ribosomal subunit assembly in Xenopus oocytes.

Abstract

Structural requirements of 5S rRNA for nuclear transport and RNA-protein interactions have been studied by analyzing the behavior of oocyte-type 5S rRNA and of 31 different in vitro-generated mutant transcripts after microinjection into the cytoplasm of Xenopus oocytes. Experiments reveal that the sequence and secondary and/or tertiary structure requirements of 5S rRNA for nuclear transport, storage in the cytoplasm as 7S ribonucleoprotein particles, and assembly into 60S ribosomal subunits are complex and nonidentical. Elements of loops A, C, and E, helices II and V, and bulged and hinge nucleotides in the central domain of 5S rRNA carry the essential information for these functional activities. Assembly of microinjected 5S rRNA into 60S ribosomal subunits was shown to occur in the nucleus; thus, the first requirement for subunit assembly is nuclear targeting. The inhibitory effects of ATP depletion, wheat germ agglutinin, and chilling on the nuclear import of 5S rRNA indicate that it crosses the nuclear envelope through the nuclear pore complex by a pathway similar to that used by karyophilic proteins.The orchestration of ribosome biogenesis in eukaryotic cells is a process that requires transfer of macromolecules into and out of the nucleus. In Xenopus oocytes, 5S rRNA is shuttled between the nuclear and cytoplasmic compartments of the oocyte during different stages of oogenesis in a complex pathway involving different protein associations. In previtellogenic oocytes, 5S rRNA is synthesized before other components of ribosomes are available, is exported from the nucleus, and stored in the cytoplasm as 7S ribonucleoprotein particles (RNPs) (5S rRNA complexed with transcription factor IIIA [TFIIIA]) or as 42S RNPs (5S rRNA complexed with other nonribosomal proteins and tRNA). During vitellogenesis, the 5S rRNA is released from storage and a 5S rRNA-ribosomal protein L5 complex, which is a precursor to assembly into the 60S large ribosomal subunit, forms (reference 3 and references therein). We are interested in the mechanisms that govern the subcellular trafficking of 5S rRNA within the oocyte, particularly the requirements for the mobilization of stored 5S rRNA during ribosome assembly.There nuclear RNAs (50), mRNA (16), 40S and 60S ribosomal subunits (6, 38), and 5S rRNA (24) occurs in a manner consistent with a mediated process. Analysis of the nuclear transport of U small nuclear RNAs has multiple defined, kinetically distinct targeting pathways (27,47,48). Nuclear transport of different classes of RNA may thus involve targeting to the pore complex by different cytoplasmic receptors and then translocation into the nucleus by the same pore complex-mediated mechanism. Specific RNA structures have been implicated as requirements for both nuclear import and export (5,22,32,33,67,75). Translocation of RNA molecules across the nuclear envelope may also require interaction with specific proteins (28,31,33,43,62). RNA-protein interactions are important for many regulatory processes. There is growing evidence that R...

Saturation Mutagenesis of 5S rRNA in Saccharomyces cerevisiae

“…In eukaryotic cells, 5S rRNA is synthesized in the nucleolus by RNA polymerase III, processed into its mature form, and then imported into the nucleus, where it associates with ribosomal protein L5. The 5S-L5 ribonuclear particle is reimported into the nucleolus, where it is assembled into the central protuberance as one of the last steps in the biogenesis of the 60S subunit (1,3,11,12). The central protuberance lies opposite the head of the small subunit, and chemical probing and X-ray crystallographic analyses provide evidence to support the notion that 5S rRNA is in close contact with this subunit (56, 66).…”

Two distinct structural elements of 5S rRNA are needed for its import into human mitochondria

“…(A) Human 5S rRNA secondary structure (mfold, corrected manually according to structural studies published). Sites of protein binding are colored (Baudin et al 1991;Chow et al 1992;White et al 1992;Allison et al 1993;Wimberly et al 1993;Szymanski et al 2000;Huber et al 2001;Lu et al 2003;Zuker 2003). (B) The overall mutation map of the human 5S rRNA obtained in this work.…”

A possible role for the 60-kD Ro autoantigen in a discard pathway for defective 5S rRNA precursors.

“…In yeast, expression of a variety of mutant 5S rRNAs did not affect growth rates significantly, even when ~<80% of the ribosomal 5S rRNA was replaced by mutant RNA (Van Ryk et al 1990. In Xenopus, Allison et al (1993) injected 31 in vitro-synthesized 5S rRNA mutants into oocytes. Surprisingly, only four of these mutants were completely defective in ribosome assembly.…”