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

Abstract

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. In eukaryotes, 79 ribosomal proteins associate with ribosomal RNA (rRNA) to produce 40S and 60S ribosomal subunits (Woolford and Warner 1991). Three of the four rRNAs in mature ribosomes are derived from the 35S-45S rRNA precursor (pre-rRNA) transcribed by RNA polymerase I, while the fourth rRNA, 5S rRNA, is transcribed from separate genes by RNA polymerase III. The 35S-45S primary transcript is packaged into a 90S ribonucleoprotein particle (RNP), together with a subset of assembly factors and ribosomal proteins. Subsequent steps trigger folding, modification, and processing of prerRNAs and association of additional assembly factors and ribosomal proteins in 43S and 66S assembly intermediates. These pre-rRNPs undergo further maturation in the nucleolus, nucleoplasm, and then cytoplasm to form functional 40S and 60S ribosomal subunits, respectively ( Fig. 1A; FromontRacine et al. 2003;Raué 2003;Granneman and Baserga 2004). Preribosomal particles in the assembly pathway are distinguished by the presence of successive prerRNA processing intermediates (Fig. 1A). However, it is not clear into which of the consecutive preribosomes 5S rRNA and each ribosomal protein are incorporated, which assembly factors are required to recruit these molecules, or how they do so. Furthermore, the mechanisms by which constituents of nascent ribosomes facilitate folding, processing, and modification of pre-rRNAs remain elusive.5S rRNA is essential for maturation of preribosomes and for the function of mature ribosomes (Van Ryk et al. 1992;Dechampesme et al. 1999;Kiparisov et al. 2005). Steitz and coworkers defined a pathway of assembly of 5S rRNA into ribosomes in HeLa cells. Newly synthesized 5S pre-rRNA binds transiently to the La protein (Rinke and Steitz 1982;Yoo and Wolin 1994). Following 3Ј-end maturation, 5S rRNA binds to ribosomal protein rpL5, then assembles into ribosomes (...

60S ribosome biogenesis requires rotation of the 5S ribonucleoprotein particle

“…This unexpected finding is consistent with the incorporation of a preformed 5S RNP into the pre-60S subunit 11 . Moreover, the higher resolution cryo-EM map allowed us to assign, in addition to the previously identified biogenesis factors Arx1 (refs 19,22) and eIF6 (refs 18,20), the precise locations of Rlp7, Rlp24, Mrt4, Nog1 and Rsa4 on the Arx1 particle.…”

“…In contrast, 5S rRNA is transcribed separately by RNA polymerase III, and its precursor matures to the 5S rRNA independently from the pre-35S RNA processing pathway 9,10 . Subsequently, 5S rRNA recruits two r-proteins, L5 and L18 (previously called Rpl11 and Rpl5 in yeast, respectively), to form the 5S ribonucleoprotein (RNP) particle, which becomes incorporated en bloc into the evolving pre-60S subunit [11][12][13] . In the mature 80S ribosome, the 5S rRNA is located within the central protuberance of the 60S subunit, where it is involved in intersubunit interactions, mediated by a number of protein-RNA and RNA-RNA contacts 14,15 .…”

Identification of mutations in the ribosomal protein L5 (RPL5) and ribosomal protein L11 (RPL11) genes in Czech patients with Diamond-Blackfan anemia

“…Another recent report indicates that processing of 27SB pre-rRNA requires a subcomplex consisting of Rpf2, Rrs1, 5S rRNA, RPL5, and RPL11, and each of the four protein components is required for production of 60S ribosomal subunits [Zhang et al, 2007]. It seems, therefore, likely that RPL11 similarly to RPL5 takes part in 27SB pre-rRNA processing as well.…”

A Puzzle of Life: Crafting Ribosomal Subunits

“…Formation of the 5S RNP is facilitated by the transport adaptor Syo1, which couples nuclear import of uL5 and uL18 with 5S rRNA association [90,91]. The subsequent incorporation of the 5S RNP module involves the Rpf2-Rrs1 heterodimer and has already occurred within the Nsa1-defined particle [25,86,[92][93][94] (Figure 2K). However, the exact timing and the mechanistic details of the initial 5S RNP docking to the pre-60S ribosome remain to be determined.…”