Yeast ribosomal proteins L4, L17, L20, and L25 exhibit different binding characteristics for the yeast 35S precursor rRNA

Yeh, Lee Chuan C; Lee, John C.

doi:10.1016/s0167-4781(98)00202-4

Cited by 10 publications

(8 citation statements)

References 59 publications

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“…For better separation of primer extension products ending at 2875–2913 nt refer to Supplementary Figure S4A. ( B ) Locations of nucleotides that are more modified in the absence of Has1 (blue circles) are displayed on the 5.8S/25S rRNA secondary structure (domain I) (http://www.rna.ccbb.utexas.edu/) and the predicted hairpin structure of ITS2 (53). The chemical modification pattern in wild-type cells (green circles) corresponds to the predicted secondary structure of domain I and ITS2.…”

Section: Resultsmentioning

confidence: 99%

Has1 regulates consecutive maturation and processing steps for assembly of 60S ribosomal subunits

Dembowski

Kuo

Woolford

2013

Nucleic Acids Research

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Ribosome biogenesis requires ∼200 assembly factors in Saccharomyces cerevisiae. The pre-ribosomal RNA (rRNA) processing defects associated with depletion of most of these factors have been characterized. However, how assembly factors drive the construction of ribonucleoprotein neighborhoods and how structural rearrangements are coupled to pre-rRNA processing are not understood. Here, we reveal ATP-independent and ATP-dependent roles of the Has1 DEAD-box RNA helicase in consecutive pre-rRNA processing and maturation steps for construction of 60S ribosomal subunits. Has1 associates with pre-60S ribosomes in an ATP-independent manner. Has1 binding triggers exonucleolytic trimming of 27SA3 pre-rRNA to generate the 5′ end of 5.8S rRNA and drives incorporation of ribosomal protein L17 with domain I of 5.8S/25S rRNA. ATP-dependent activity of Has1 promotes stable association of additional domain I ribosomal proteins that surround the polypeptide exit tunnel, which are required for downstream processing of 27SB pre-rRNA. Furthermore, in the absence of Has1, aberrant 27S pre-rRNAs are targeted for irreversible turnover. Thus, our data support a model in which Has1 helps to establish domain I architecture to prevent pre-rRNA turnover and couples domain I folding with consecutive pre-rRNA processing steps.

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Section: Resultsmentioning

confidence: 99%

Has1 regulates consecutive maturation and processing steps for assembly of 60S ribosomal subunits

Dembowski

Kuo

Woolford

2013

Nucleic Acids Research

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“…Although the differences between these two 60S localization assays may seem minor, they have the potential to identify a different set of factors required for ribosomal subunit assembly and export. Because Rpl25 binds directly to the rRNA, it is likely to be buried within the 60S ribosomal subunit (El-Baradi et al, 1984, 1987Yeh and Lee, 1998). Rpl11 binds later in ribosome assembly and appears to be located at the surface of the 60S ribosomal subunit (Tsay et al, 1994).…”

Section: Discussionmentioning

confidence: 99%

Factors Affecting Nuclear Export of the 60S Ribosomal Subunit In Vivo

Stage-Zimmermann

Schmidt

Silver

2000

MBoC

132

121

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In Saccharomyces cerevisiae, the 60S ribosomal subunit assembles in the nucleolus and then is exported to the cytoplasm, where it joins the 40S subunit for translation. Export of the 60S subunit from the nucleus is known to be an energy-dependent and factor-mediated process, but very little is known about the specifics of its transport. To begin to address this problem, an assay was developed to follow the localization of the 60S ribosomal subunit in S. cerevisiae. Ribosomal protein L11b (Rpl11b), one of the ϳ45 ribosomal proteins of the 60S subunit, was tagged at its carboxyl terminus with the green fluorescent protein (GFP) to enable visualization of the 60S subunit in living cells. A panel of mutant yeast strains was screened for their accumulation of Rpl11b-GFP in the nucleus as an indicator of their involvement in ribosome synthesis and/or transport. This panel included conditional alleles of several rRNA-processing factors, nucleoporins, general transport factors, and karyopherins. As predicted, conditional alleles of rRNAprocessing factors that affect 60S ribosomal subunit assembly accumulated Rpl11b-GFP in the nucleus. In addition, several of the nucleoporin mutants as well as a few of the karyopherin and transport factor mutants also mislocalized Rpl11b-GFP. In particular, deletion of the previously uncharacterized karyopherin KAP120 caused accumulation of Rpl11b-GFP in the nucleus, whereas ribosomal protein import was not impaired. Together, these data further define the requirements for ribosomal subunit export and suggest a biological function for KAP120. INTRODUCTIONAlthough eukaryotic ribosomes function in the cytoplasm, the synthesis, processing, and assembly of the ribosomal subunits in Saccharomyces cerevisiae and higher eukaryotes occur in the nucleolus. The entire ribosome is composed of four rRNA species and ϳ75 ribosomal proteins (r-proteins) distributed between two subunits. The 18S, 5.8S, and 25S rRNAs are derived from a single 35S rRNA precursor that is synthesized by RNA polymerase I and then processed by a series of endonucleolytic and exonucleolytic cleavages (reviewed by Kressler et al., 1999;Venema and Tollervey, 1999). The 5S rRNA is synthesized separately by RNA polymerase III and associates with the 60S preribosomal subunit early in assembly. The mature 40S ribosomal subunit contains the 18S rRNA and ϳ32 r-proteins, whereas the 60S subunit is composed of the 5S, 5.8S, and 25S rRNAs and ϳ45 r-proteins. Proper assembly of each ribosomal subunit requires the coordination of several events, including the synthesis and import of r-proteins, the synthesis and processing of rRNA, and the concomitant assembly of r-proteins into the preribosomal subunits. Although a pathway for 35S rRNA maturation has been well defined through both genetic and biochemical approaches (reviewed by Kressler et al., 1999;Venema and Tollervey, 1999), less is known about the association of r-proteins with the rRNA and the export of the assembled subunits out of the nucleus.All nucleocytoplasmic transport occurs ...

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“…It has also been shown that three large-subunit r-proteins on mature 60S subunits can exchange in vivo, and the exchangeability of Rpl10p/Qsr1p, in particular, which is required for 60S to 40S subunit joining, suggests the possibility of an additional translational regulatory mechanism (46,209). Due to the lack of in vitro reconstitution assays for eukaryotic ribosomes, the abovementioned studies could only be complemented by approaches that examined the abilities of the different r-proteins to either bind to pre-rRNA molecules in vitro (204) or dissociate from purified ribosomal particles by increasing concentration of ions (52,106). The affinity of r-proteins for pre-rRNAs and the release of r-proteins by increasing concentration of ions are in general agreement with the order of their association with the preribosomal particles during in vivo ribosomal subunit assembly.…”

Section: Factors Involved In Ribosome Assembly?mentioning

confidence: 99%

Protein trans-Acting Factors Involved in Ribosome Biogenesis in Saccharomyces cerevisiae

Kressler

Linder

Cruz

1999

Molecular and Cellular Biology

342

343

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2The synthesis of ribosomes is one of the major cellular activities, and in eukaryotes, it takes place primarily, although not exclusively, in a specialized subnuclear compartment termed the nucleolus (125, 155). There, the rRNA genes are transcribed as precursors (pre-rRNAs), which undergo processing and covalent modification. Maturation of pre-rRNAs is intimately linked to their assembly with the ribosomal proteins (r-proteins). These processes depend on various cis-acting elements (6, 188), and they require a large number of nonribosomal protein trans-acting factors (97,174,193). Experimental evidence suggests that the basic outline of ribosome synthesis is conserved throughout eukaryotes. However, most of our knowledge comes from the combination of molecular genetic and biochemical approaches in the yeast Saccharomyces cerevisiae. This minireview is aimed at giving an insight into the functions of the many protein trans-acting factors involved in ribosome biogenesis in S. cerevisiae. PRE-rRNA PROCESSING AND RIBOSOME ASSEMBLY PATHWAYSIn S. cerevisiae, the large 60S ribosomal subunits are composed of 46 r-proteins and three rRNA species (5S, 5.8S, and 25S) while the small 40S ribosomal subunits contain 32 rproteins and the 18S rRNA (142,201). Three of the four rRNAs (18S, 5.8S, and 25S) are transcribed as a single large pre-rRNA by RNA polymerase I (RNA pol I), whereas the fourth rRNA (5S) is independently transcribed as a pre-rRNA by RNA pol III (201). All four rRNAs are encoded by a 9.1-kb rDNA unit, which is repeated 100 to 200 times on the long arm of chromosome XII (Fig. 1A). In the 35S pre-rRNA, which is the longest detectable precursor, the mature rRNA sequences are separated by two internal transcribed spacer (ITS) sequences, ITS1 and ITS2, and flanked by two external transcribed spacer (ETS) sequences, a 5Ј ETS and a 3Ј ETS (Fig. 1). The 35S pre-rRNA differs from the primary RNA pol I transcript at its 3Ј end because transcription termination maps to nucleotide position ϩ210 relative to the 3Ј end of the mature 25S rRNA, while the 35S pre-rRNA is extended by 7 to 10 nucleotides (78,187,189). Maturation of the 35S pre-rRNA, which contains 10 known processing sites, is a multistep pathway that requires many different trans-acting factors (Fig. 1B and its legend) (97,174,193). Processing of the pre-5S rRNA is independent of 35S pre-rRNA maturation and kinetically faster than formation of mature 18S, 5.8S, and 25S rRNAs (146). The 5Ј end of the mature 5S rRNA corresponds to that of the primary transcript, whereas the 3Ј end is processed from a pre-5S rRNA that is extended by 7 to 13 nucleotides (141). Many specific nucleotides within the rRNA also undergo, mainly shortly after transcription, covalent modification. These modifications include isomerization of uridine to pseudouridine (⌿) by base rotation (45 modified nucleotides), methylation of the 2Ј-hydroxyl group of sugar residues (2Ј-O-ribose methylation; 55 modified nucleotides), and base methylation (about 10 modified nucleotides) (9,21,84,120,135...

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Yeast ribosomal proteins L4, L17, L20, and L25 exhibit different binding characteristics for the yeast 35S precursor rRNA

Cited by 10 publications

References 59 publications

Has1 regulates consecutive maturation and processing steps for assembly of 60S ribosomal subunits

Has1 regulates consecutive maturation and processing steps for assembly of 60S ribosomal subunits

Factors Affecting Nuclear Export of the 60S Ribosomal Subunit In Vivo

Protein trans-Acting Factors Involved in Ribosome Biogenesis in Saccharomyces cerevisiae

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