Paralog-Specific Functions ofRPL7AandRPL7BMediated by Ribosomal Protein or snoRNA Dosage inSaccharomyces cerevisiae

Abstract

Most ribosomal proteins in Saccharomyces cerevisiae are encoded by two paralogs that additively produce the optimal protein level for cell growth. Nonetheless, deleting one paralog of most ribosomal protein gene pairs results in a variety of phenotypes not observed when the other paralog is deleted. To determine whether paralog-specific phenotypes associated with deleting RPL7A or RPL7B stem from distinct functions or different levels of the encoded isoforms, the coding region and introns of one paralog, including an intron-embedded snoRNA (small nucleolar RNA) gene, were exchanged with that of the other paralog. Among mutants harboring a single native or chimeric RPL7 allele, expression from the RPL7A locus exceeded that from the RPL7B locus, and more Rpl7a was expressed from either locus than Rpl7b. Phenotypic differences in tunicamycin sensitivity, ASH1 mRNA localization, and mobility of the Ty1 retrotransposon were strongly correlated with Rpl7 and ribosome levels, but not with the Rpl7 or snoRNA isoform expressed. Although Ty1 RNA is cotranslationally localized, depletion of Rpl7 minimally affected synthesis of Ty1 Gag protein, but strongly influenced Ty1 RNA localization. Unlike the other processes studied, Ty1 cDNA accumulation was influenced by both the level and isoform of Rpl7 or snoRNA expressed. These cellular processes had different minimal threshold values for Rpl7 and ribosome levels, but all were functional when isoforms of either paralog were expressed from the RPL7A locus or both RPL7 loci. This study illustrates the broad range of phenotypes that can result from depleting ribosomes to different levels.

Does functional specialization of ribosomes really exist?

“…A similar case is seen with Rpl7/uL30, where phenotypes that appear paralog-specific are actually responsive to Rpl7 dose (Palumbo et al 2017). Deletion of Rpl7A leads to specific phenotypes including slow growth, tunicamycin resistance and defects in bud tip localization of the ASH1 mRNA.…”

“…An important example for such effects from ribosome numbers are recent observations that knockdown of two different RPs had highly overlapping effects on gene-specific translation in hematopoetic progenitor cells (Khajuria et al 2018). Similarly, as detailed below, several studies on differential effects from knockout of RP paralogs in yeast are most simply consistent with effects arising from reduced ribosome numbers (Rotenberg et al 1988;Watanabe et al 1993;Zinn et al 1994;Strittmatter et al 2006;Palumbo et al 2017).…”

“…Genuth and Barna 2018a; Guo 2018]) provide compelling evidence for ribosome heterogeneity, demonstrating that these differences in ribosome composition have functional consequences is challenging. This is because most manipulations of ribosomal composition also change ribosome number (Rotenberg et al 1988;Choesmel et al 2007;Li et al 2009;Bhattacharya et al 2010;McIntosh et al 2011;Steffen et al 2012;Ferretti et al 2017;Palumbo et al 2017;Cheng et al 2018a;Segev and Gerst 2018). Furthermore, changing the expression of one paralog often affects the expression of the other.…”

“…The "ribosome concentration hypothesis" (Mills and Green 2017) is important to consider as ribosome numbers are reduced in many experimental systems used to study specialized ribosomes (Rotenberg et al 1988;Bhattacharya et al 2010;McIntosh et al 2011;Vesper et al 2011;Steffen et al 2012;Thompson et al 2016;Ferretti et al 2017;Palumbo et al 2017;Cheng et al 2018a;Segev and Gerst 2018), making these studies particularly vulnerable to this confounding variable, and demonstrating the importance of rigorously controlling for effects arising from ribosome numbers. An important example for such effects from ribosome numbers are recent observations that knockdown of two different RPs had highly overlapping effects on gene-specific translation in hematopoetic progenitor cells (Khajuria et al 2018).…”

Translational regulation in response to stress in Saccharomyces cerevisiae

“…For example, the uL30 paralogues each contain introns encoding different small nucleolar RNAs that both guide methylation of 25S rRNA at A807 (Samarsky & Fournier, 1999). A more recent study provided further evidence that differences in the loci, rather than in the proteins themselves, contribute to the paralogue-specific functions for this pair (Palumbo et al, 2016), suggesting that, although the proteins have different physiological roles, their actual structure and function within ribosomes may be equivalent.…”