The amino terminal domain from Mrt4 protein can functionally replace the RNA binding domain of the ribosomal P0 protein

In Saccharomyces cerevisiae, the Mrt4 protein is a component of the ribosome assembly machinery that shares notable sequence homology to the P0 ribosomal stalk protein. Here, we show that these proteins can not bind simultaneously to ribosomes and moreover, a chimera containing the first 137 amino acids of Mrt4 and the last 190 amino acids from P0 can partially complement the absence of the ribosomal protein in a conditional P0 null mutant. This chimera is associated with ribosomes isolated from this strain when grown under restrictive conditions, although its binding is weaker than that of P0. These ribosomes contain less P1 and P2 proteins, the other ribosomal stalk components. Similarly, the interaction of the L12 protein, a stalk base component, is affected by the presence of the chimera. These results indicate that Mrt4 and P0 bind to the same site in the 25S rRNA. Indeed, molecular dynamics simulations using modelled Mrt4 and P0 complexes provide further evidence that both proteins bind similarly to rRNA, although their interaction with L12 displays notable differences. Together, these data support the participation of the Mrt4 protein in the assembly of the P0 protein into the ribosome and probably, that also of the L12 protein

Role and dynamics of the ribosomal protein P0 and its related trans-acting factor Mrt4 during ribosome assembly in Saccharomyces cerevisiae

Mrt4 is a nucleolar component of the ribosome assembly machinery that shares notable similarity and competes for binding to the 25S rRNA GAR domain with the ribosomal protein P0. Here, we show that loss of function of either P0 or Mrt4 results in a deficit in 60S subunits, which is apparently due to impaired rRNA processing of 27S precursors. Mrt4, which shuttles between the nucleus and the cytoplasm, defines medium pre-60S particles. In contrast, P0 is absent from medium but present in late/cytoplasmic pre-60S complexes. The absence of Mrt4 notably increased the amount of P0 in nuclear Nop7–TAP complexes and causes P0 assembly to medium pre-60S particles. Upon P0 depletion, Mrt4 is relocated to the cytoplasm within aberrant 60S subunits. We conclude that Mrt4 controls the position and timing of P0 assembly. In turn, P0 is required for the release of Mrt4 and exchanges with this factor at the cytoplasm. Our results also suggest other P0 assembly alternatives

Principles of mRNA transport in yeast

mRNA localization and localized translation is a common mechanism by which cellular asymmetry is achieved. In higher eukaryotes the mRNA transport machinery is required for such diverse processes as stem cell division and neuronal plasticity. Because mRNA localization in metazoans is highly complex, studies at the molecular level have proven to be cumbersome. However, active mRNA transport has also been reported in fungi including Saccharomyces cerevisiae, Ustilago maydis and Candida albicans, in which these events are less difficult to study. Amongst them, budding yeast S. cerevisiae has yielded mechanistic insights that exceed our understanding of other mRNA localization events to date. In contrast to most reviews, we refrain here from summarizing mRNA localization events from different organisms. Instead we give an in-depth account of ASH1 mRNA localization in budding yeast. This approach is particularly suited to providing a more holistic view of the interconnection between the individual steps of mRNA localization, from transcriptional events to cytoplasmic mRNA transport and localized translation. Because of our advanced mechanistic understanding of mRNA localization in yeast, the present review may also be informative for scientists working, for example, on mRNA localization in embryogenesis or in neurons

Rational Extension of the Ribosome Biogenesis Pathway Using Network-Guided Genetics

Gene networks are an efficient route for associating candidate genes with biological processes. Here, networks are used to discover more than 15 new genes for ribosomal subunit maturation, rRNA processing, and ribosomal export from the nucleus

Assembly factors Rpf2 and Rrs1 recruit 5S rRNA and ribosomal proteins rpL5 and rpL11 into nascent ribosomes.

More than 170 proteins are necessary for assembly of ribosomes in eukaryotes. However, cofactors that function with each of these proteins, substrates on which they act, and the precise functions of assembly factors--e.g., recruiting other molecules into preribosomes or triggering structural rearrangements of pre-rRNPs--remain mostly unknown. Here we investigated the recruitment of two ribosomal proteins and 5S ribosomal RNA (rRNA) into nascent ribosomes. We identified a ribonucleoprotein neighborhood in preribosomes that contains two yeast ribosome assembly factors, Rpf2 and Rrs1, two ribosomal proteins, rpL5 and rpL11, and 5S rRNA. Interactions between each of these four proteins have been confirmed by binding assays in vitro. These molecules assemble into 90S preribosomal particles containing 35S rRNA precursor (pre-rRNA). Rpf2 and Rrs1 are required for recruiting rpL5, rpL11, and 5S rRNA into preribosomes. In the absence of association of these molecules with pre-rRNPs, processing of 27SB pre-rRNA is blocked. Consequently, the abortive 66S pre-rRNPs are prematurely released from the nucleolus to the nucleoplasm, and cannot be exported to the cytoplasm.</p