Rea1, a Dynein-related Nuclear AAA-ATPase, Is Involved in Late rRNA Processing and Nuclear Export of 60 S Subunits

Abstract

Rea1, the largest predicted protein in the yeast genome, is a member of the AAA ؉ family of ATPases and is associated with pre-60 S ribosomes. Here we report that Rea1 is required for maturation and nuclear export of the pre-60 S subunit. Rea1 exhibits a predominantly nucleoplasmic localization and is present in a late pre-60 S particle together with members of the Rix1 complex. To study the role of Rea1 in ribosome biogenesis, we generated a repressible GAL::REA1 strain and temperaturesensitive rea1 alleles. In vivo depletion of Rea1 results in the significant reduction of mature 60 S subunits concomitant with defects in pre-rRNA processing and late pre-60 S ribosome stability following ITS2 cleavage and prior to the generation of mature 5.8 S rRNA. Strains depleted of the components of the Rix1 complex (Rix1, Ipi1, and Ipi3) showed similar defects. Using an in vivo 60 S subunit export assay, a strong accumulation of the large subunit reporter Rpl25-GFP (green fluorescent protein) in the nucleus and at the nuclear periphery was seen in rea1 mutants at restrictive conditions.The synthesis of ribosomes is one of the major and most energy-consuming processes in the cell. In Saccharomyces cerevisiae, ribosome biogenesis begins in the nucleolus with the transcription of two rRNA precursors, the 35 S and the pre-5 S RNA, by RNA polymerases I and III, respectively. The 35 S pre-rRNA contains the sequences for the mature 18 S, 5.8 S, and 25 S rRNAs, two external transcribed spacers (ETS) 1 and two internal transcribed spacers (ITS). During the maturation process, the pre-rRNA has to undergo a number of modifications and is subjected to cleavages and trimming events. At least 170 accessory proteins including putative RNA helicases, endo-and exonucleases, and putative GTPases and AAAATPases as well as small nucleolar ribonucleoprotein particles are involved in the maturation of rRNA and its assembly into ribosomal subunits (1, 2).Concomitant with rRNA processing, ribosomal and non-ribosomal proteins are assembled on the pre-35 S rRNA, giving rise to a large 90 S pre-ribosomal particle (see Fig. 6B) (3, 4). The initial cleavages at sites A 0 -A 2 separate the two subunits. The pre-40 S subunit is exported relatively rapidly to the cytoplasm, where it undergoes further processing. In contrast, the maturation of the large subunit continues in the nucleoplasm with recruitment of 60 S-specific biogenesis factors and further processing of the 27 S pre-rRNA. This includes the late cleavage and processing in the ITS2 region, which generates mature 5.8 S and 25 S rRNA.In the last few years, the maturation of 40 S and 60 S pre-ribosomes has been extensively analyzed by purification of pre-ribosomal particles (5-10). Interestingly, a large number of non-ribosomal proteins were identified in pre-60 S particles without an assigned function in RNA metabolism. In contrast to the pre-40 S particles, the nascent 60 S particles contain several putative GTPases and AAA-type ATPases (2, 11).To understand the events of ribosome biogenesis, w...

The MIDASIN and NOTCHLESS genes are essential for female gametophyte development in Arabidopsis thaliana

“…The identification of both NLE/RSA4 and MDN1/ REA1 in 60S ribosomal subunit biogenesis in yeast (Galani et al, 2004;Nissan et al, 2002) and their interaction in plants (Chantha and Matton, 2007) and in yeast (Ulbrich et al, 2009) (Chantha et al, 2006). Moreover, the semisterility phenotype was maintained in the following generations.…”

“…In yeast, both MDN1/ REA1 and NLE/RSA4 are essential since mutations in these genes lead to cell lethality while depletion of both proteins causes a significant slow growth phenotype (de la Cruz et al, 2005;Galani et al, 2004). Underexpression of ScNLE in S. chacoense by RNA interference was previously shown to cause a pleiotropic phenotype during plant sporophytic development, the major defect being the production of smaller organs due to reduced cell proliferation and cell enlargement (Chantha and Matton, 2007).…”

“…Although NLE/RSA4 and MDN1/REA1 are involved in the same cellular process in yeast, they do not accomplish the same function during 60S subunit biogenesis and their respective function may not be equally essential. In yeast, while NLE/RSA4 represents one of the few trans-acting factors involved in all the maturation steps of the 60S ribosomal subunit, from early on in the nucleolus to the final maturation steps in the cytoplasm (Nissan et al, 2002), MDN1/REA1 is only involved in late nucleoplasmic pre-60S complexes that are close to their export to the cytoplasm (Galani et al, 2004). Another explanation would be that the function of NLE/RSA4 and/or MDN1/REA1 may not be restricted to 60S subunit biogenesis, as was determined for other 60S trans-acting factors (Tschochner and Hurt, 2003).…”

Interactions among Ytm1, Erb1, and Nop7 Required for Assembly of the Nop7-Subcomplex in Yeast Preribosomes

“…At least fourteen different subcomplexes have been identified from preribosomal particles in yeast, revealing the modular nature of ribosome assembly (Harnpicharnchai et al, 2001;Du and Stillman, 2002;Granneman et al, 2003;Dosil and Bustelo, 2004;Galani et al, 2004;Krogan et al, 2004;Miles et al, 2005;Karbstein and Doudna, 2006;Rashid et al, 2006;Schäfer et al, 2006;Rosado et al, 2007;Rudra et al, 2007;Zhang et al, 2007). Interactions among components of a subcomplex, such as the Nop7-or PeBoW-subcomplex (Lapik et al, 2004;Miles et al, 2005), the Rpf2-subcomplex (Morita et al, 2002;Miyoshi et al, 2004;Nariai et al, 2005;Zhang et al, 2007), or the Arx1-subcomplex (Lebreton et al, 2006), have been inferred from indirect yeast two-hybrid experiments in vivo, GST pulldown assays in vitro, or by assays for genetic interactions.…”

Ribosome Biogenesis in the YeastSaccharomyces cerevisiae

“…These include the AAA ATPase Rea1 and the Rix1, Ipi1, and Ipi3 subcomplex (Galani et al 2004). Removal of Ytm1, Rsa4, or other assembly factors from preribosomes by Rea1 might be necessary to provide access for nucleases or to properly organize the RNP environment for processing of the 39 end of 7S pre-rRNA.…”