Introduction
In eukaryotes, 5’→3’ decay plays a crucial role in controlling RNA processing, quality, and quantity. 5’→3’ exoribonucleases (XRNs) are conserved in eukaryotes and play crucial roles in diverse aspects of mRNA and non-coding (nc) RNA processing and degradation. XRNs recognize RNAs with a 5’ monophosphate (5’P) end arising from endonucleolytic or exonucleolytic cleavage or decapping, and trim the RNA processively in the 5’→3’ direction (Nagarajan et al., 2013; Chang et al., 2011). The molecular functions of XRNs have been mainly studied in the budding yeast Saccharomyces cerevisiae , which possesses two XRNs, a mainly cytoplasmic exoribonuclease, Xrn1, and a nuclear exoribonuclease, Xrn2 (also referred to as Rat1) (Amberg et al., 1992; Heyer et al., 1995; Johnson, 1997). In yeast, Xrn1 is responsible for cytoplasmic deadenylation-dependent mRNA 5′→3′ exonucleolytic decay (Parker & Song, 2004, Nagarajan et al., 2013). Indeed, the demonstrated activity as 5’→3’ exoribonuclease (Larimer & Stevens, 1990, Larimer et al., 1992) is the reason for its Xrn1 (ex or ibon uclease1 ) name. Xrn1 5′→3′ exonucleolytic activity on mRNAs is fundamental for the decay of both correct and defective molecules, provided that they have a 5’P end caused by either decapping or endonucleolytic cleavage (Parker & Song, 2004, Nagarajan et al., 2013). Xrn1p can also hydrolyze NAD-capped mRNAs (deNADase activity) and, afterwards, exonucleolytically degrade them processively (4). Its enzymatic activity is highly processive and can even act co-translationally on ribosome-associated mRNAs. In this context, Xrn1 trails the last translating ribosome (Pelechano et al, 2015) with which it physically interacts specifically (Tesina et al., 2019). Xrn1 plays additional roles in mRNA processing and quality control, rRNA processing, tRNA quality and ncRNA decay (Parker & Song, 2004; Nagarajan et al., 2013; Langeberg et al., 2020; Sharma et al., 2022).
Both 5’→3’ exoribonucleases are partially homologous (Nagarajan et al., 2013; Kenna et al., 1993) and have been shown to be functionally interchangeable, although they were thought to be restricted to specific cellular compartments: Xrn1 in the cytoplasm and Rat1 in the nucleus (Johnson, 1997). RAT1 is an essential gene, whereas XRN1is not (Johnson, 1997). Ectopic nuclear localization of Xrn1 by the addition of an SV40 nuclear location signal (NLS) can rescue rat1lethality, whereas cytoplasmic Rat1, lacking its NLS, can rescuexrn1 ski2 lethality (Johnson, 1997). SKI2 encodes a co-factor of the 3’→5’ RNA exosome, which is essential for its cytoplasmic function (Johnson & Kolodner, 1995). The enzymatic mechanism of Rat1 is assumed to be processive and similar to that of Xrn1 because it shares the active site and shows extensive conservation around it (Nagarajan et al., 2013; Basu et al., 2021). Rat1 is also a deNADing enzyme similar to Xrn1 (Sharma et al., 2022). Rat1 performs important nuclear activities related to RNA metabolism, including rRNA and snoRNA processing, as well as poly-A+ dependent and independent mRNA transcription termination (Kim et al., 2004). The existence of both nuclear (Rat1/Xrn2 family) and cytoplasmic (Xrn1 family) 5’→3’ exoribonucleases is common in a variety of studied eukaryotes (Chang et al., 2011; Han et al., 2023) and suggests that functional interchangeability extends across species (reviewed in Nagarajan et al., 2013).
Rat1 apparently lacks cytoplasmic functions (Johnson, 1997), whereas it has been shown for Xrn1 that, albeit predominantly cytoplasmic (Johnson, 1997; Haimovich et al., 2013), it also plays important nuclear functions, such as rRNA processing and degradation of defective RNAs (Nagarajan et al., 2013). Xrn1 also acts as a general transcription activator, forming a complex with other decay factors (Haimovich et al., 2013; Medina et al., 2014). The proper cytoplasmic function of Xrn1p requires its shuttling between the cytoplasm and nucleus (Pérez-Ortín & Chavez, 2022), which is made possible by the recently discovered existence of two NLSs (Chattopadhyay et al., 2022). Budding yeast Xrn1 is a very long protein with 1528 amino acids compared to the shorter Rat1 (1006 amino acids). The larger size of Xrn1 is due to an extended C-terminal (Cterm) segment that is absent in Rat1 (Chang et al., 2012), which is a basic domain that is not essential for most in vivofunctions and is toxic when overexpressed (Page et al., 1998). This feature and the relatively low homology between budding yeast Xrn1 and Rat1 (40% amino acid identity) pose the question of how these two proteins can substitute one another when located in each other’s cell compartment (Johnson, 1997).
In this study, our goal was to gain insight into which of the in vivo activities of Xrn1 can be substituted with Rat1. We studied two yeast strains that express RAT1 with defective NLS, the product of which is cytoplasmic (cRat1). We found that cRat1 partially complements many of the natural features of Xrn1, including co-translational mRNA decay. This complementation is not improved by adding Xrn1 Cterm to cRat1 despite the fact that this construction was previously shown to complement other differences in cRat1 activity (Blasco-Moreno et al., 2019). These results support the idea that both 5’→3’ exonucleases have functionally redundant activities, but have acquired some unique features, including their cellular localization, that seem to optimize their function and possibly their cooperation. Our results suggest that there are other structural disparities between the two exonucleases, potentially involving distinct binding partners, or the essential import of Xrn1 into the nucleus. These features are crucial for the complete in vivo functionality of co-translational mRNA decay..