Organellar and Secretory Ribonucleases: Major Players in Plant RNA Homeostasis

Abstract

During the last 20 years there has been an intense focus on posttranscriptional regulation of gene expression, and the role played by ribonucleases (RNases) in this process. Most research has centered on cytoplasmic and nuclear RNA decay enzymes, particularly those involved in mRNA regulation, and in proteins mediating nucleus-localized coding and noncoding RNA processing. These efforts have created a well-defined picture of RNA homeostasis in eukaryotes, including plants (Schmid and Jensen 2018; Sieburth and Vincent 2018; Towler and Newbury, 2018; Matsui et al., 2019). Thus, cytoplasmic degradation of mRNAs in plants (see Box 1), which involves deadenylation, decapping, and degradation by 3′-5′ and 5′-3′ exoribonucleases, has been extensively studied and reviewed (e.g. Zhang and Guo 2017; Sieburth and Vincent 2018; Sorenson et al., 2018; Lange et al., 2019). Similarly, different roles for the nuclear exosome and other RNases in nuclear RNA processing and decay have been established (Lange et al., 2014; Sikorska et al., 2017; Tomecki et al., 2017; Sáez-Vásquez and Delseny 2019). RNA decay mediated by small RNAs associated to RNA-induced silencing complexes and the processing and decay of small RNAs (sRNAs) have also been the focus of intense research efforts (Fukudome and Fukuhara 2017; Yu et al., 2017; Wang et al., 2019). On the other hand, RNases that do not reside in the cytoplasm or nucleus have received significant less attention, even though their activities are important for plant growth, development, and response to biotic and abiotic stimuli from the environment. As we will describe, organellar RNases are essential to maintain RNA homeostasis in chloroplasts and mitochondria. Plants also express members of the RNase T2 protein family, associated with the secretory pathway, which are involved in the maintenance of normal cellular homeostasis, with diverse roles, from stress responses to control of self-pollen rejection. This review will provide a summary of the present knowledge on these RNases, from enzymatic characterizations to biological functions, and highlight questions that should be addressed in the future. Secretory RNases are enzymes targeted to the cellular secretory system and thus localized in organelles or the extracellular space. In plants, characterized secretory RNases belong to the RNase T2 family, a group of enzymes typified by the fungal RNase T2 purified from Aspergillus oryzae (MacIntosh 2011). Enzymes from the T2 family are endoribonucleases without sequence or base specificity that degrade single-stranded RNA through a 2’,3′ cyclic phosphate intermediate, in a reaction catalyzed by two absolutely conserved His residues that define the active site (Irie 1999). It has been proposed that their enzymatic mechanism involves a two-step (transphosphorylation and hydrolysis) general acid–base catalysis (Irie 1999; Thorn et al., 2012). Interestingly, fungal enzymes can complete both steps and yield 3′ nucleotide monophosphates as final product, but bacterial enzymes end the reaction after the first step, resulting in the production of nucleotides (nt) with a 2’,3′ cyclic phosphate (Nicholson 1999; Fontaine et al., 2018). Although not many plant RNase T2 proteins have been characterized enzymatically, biochemical analyses of extracellular and vacuolar tomato enzymes indicated that plant RNase T2 enzymes generate 2’,3′ cyclic nucleotide monophosphates as primary product (Nürnberger et al., 1990; Löffler et al., 1992). The RNase T2 family is conserved in almost all eukaryotes so far analyzed, with the exception of trypanosomes and Schizosaccharomyces pombe (Shang et al., 2018; Fricker et al., 2019) and is also found in a large number of bacteria and several viruses. In most nonplant organisms, only one gene belonging to this family is found in each genome (Hillwig et al., 2009; Ambrosio et al., 2014); however, in plants, the RNase T2 protein repertoire has expanded, and individual proteins have been adapted for a variety of functions. At least four genes are found in each seed plant genome that has been sequenced (MacIntosh et al., 2010; Ramanauskas and Igić 2017), and there is evidence of frequent duplications/gene losses that resulted in different number of RNase T2 gene in different species. In addition, gene expression and functional analyses have shown that these proteins have acquired a variety of biological roles, including participation in the cellular housekeeping salvage pathway, production of tRNA-derived sRNAs, defense activities, and a central role in gametophytic self-incompatibility in several plant families (Table 1). Phylogenetic analyses allowed separation of plant RNase T2 enzymes in three clades (Igic and Kohn 2001; MacIntosh et al., 2010; Ramanauskas and Igić 2017) that define three classes of proteins roughly associated with different functions: Class I are proteins associated with a variety of stress responses and show evidence of gene duplications and gene sorting that provide large variability to this class, with variable number of class I proteins in individual species; Class II enzymes are conserved in all seed plants, in general with only one gene per genome, and their function has been related to RNA salvage; and Class III enzymes are mainly associated with self-incompatibility, although other functions have also been assigned to this class (Table 1). Historically, Class III enzymes were named S-RNases, whereas other plant T2 proteins are referred to as S-like RNases (McClure et al., 1990; Taylor et al., 1993). Class I and II proteins are found in all land plants, whereas Class III proteins are found only in core eudicots (Ramanauskas and Igić, 2017). Some plant RNase T2 proteins in the three classes have lost their catalytic activity (MacIntosh et al., 2010; Ramanauskas and Igić 2017). A monocot-specific class I subclade of proteins without RNase activity is particularly conserved (MacIntosh et al., 2010). Some of these proteins, for example OsRNS4 (Table 1), seem to have significant stress-related functions (Zheng et al., 2014), although their specific biological activity is not known. For brevity, this review will focus on two plant RNase T2 functions for which significant advances have occurred in recent years: ribosomal RNA (rRNA) turnover and tRNA processing. The presence of RNase T2 enzymes in almost all eukaryote genomes suggests that these enzymes carry out an important biological function. Phylogenetic and gene expression analyses suggested that enzymes in Class II carry out a housekeeping function in plants, and that this role is likely the ancestral function of RNase T2 enzymes (Hillwig et al., 2009; MacIntosh et al., 2010). RNS2, the Class II enzyme present in Arabidopsis (Arabidopsis thaliana), is localized mainly in the vacuole (Hillwig et al., 2011a). Characterization of rns2 mutants showed that the enzyme is necessary for normal the of and almost in the (Hillwig et al., and in et al., Plants activity showed a that housekeeping turnover of is an essential to maintain cellular homeostasis (Hillwig et al., et al., Characterization of a an active RNase the last that the of the protein a vacuolar as the protein is the vacuole and of the mutants present a to the vacuolar activity of this enzyme is essential to maintain normal cellular functions et al., 2017). Although the mechanism of ribosomal or from the cytoplasm to the vacuole is not evidence suggests that a mechanism that of the proteins necessary for the a protein for et al., 2014), in the of or to for degradation et al., RNase RNA turnover is of the salvage that normal cellular of of vacuolar RNA turnover in the rns2 in through the phosphate pathway, as a to generate for nucleotide et al., 2017). A role for RNase T2 enzymes has been in other organisms, including et al., et al., and et al., 2018). Although RNS2, and in other plants, are necessary for normal turnover and homeostasis in rns2 mutants even the is (Hillwig et al., 2011a). This that other RNases can in this turnover in the vacuole or the cytoplasm is not but rns2 plants have a is to that this RNase activities to the vacuole that to For in stress the presence of cytoplasmic complexes involved in mRNA and RNA in several ribonucleases including the and and and and has been shown that are through an in and et al., et al., 2017). It can thus be that RNases to decay in the rns2 As has been that the role in RNA salvage is the ancestral function of RNase T2 enzymes (MacIntosh 2011). their almost these enzymes are not although significant cellular and have been in plants and in RNase T2 activity (see It is that the presence of T2 enzymes provide a significant to the that in the conserved presence of these enzymes in It is important to that although the biological role has been assigned to in and of gene duplications have the function to other T2 in other species. For the Class II RNase T2 in RNase to have a role in and to to the and whereas class I enzymes are found in the tomato vacuole and be in of the salvage function in this plant and For a of and of the RNase T2 family in plants, the by Ramanauskas and Igić, is the of small RNA found in organisms, tRNA-derived small RNAs are a recent in from to and are from different of can be found in all that are likely the of in the tRNA and by of the referred as tRNA-derived and tRNA and 3′ tRNA that from processing the et al., 2019). that for these is not and to all as and and et al., Although the function of is less that of other sRNAs, their with proteins participation in posttranscriptional regulation of gene expression, with activity et al., In addition, functions have been including regulation of both and regulation of to proteins, and of mRNA by et al., 2019). the significant tRNA is not that are also conserved as as and bacteria et al., of have been characterized in a variety of plants and of individual is specific and also by biotic and abiotic stress by et al., 2018). The for production of and is et in the to in and that in These also in from and and a can be in the of et al., 2017). The found a in in the that is in the family the of these on the RNase These are found in complexes and These indicated that the are by a and in a to control other have found that the of and is only in Arabidopsis mutants et al., et al., 2017). On the other hand, et found that several and to in Arabidopsis plants a Class I RNase that this enzyme be involved in production in A of the role of plant RNase T2 enzymes in production by et RNA and in tRNA to that is not in the Arabidopsis and that protein from this are to in to and The biochemical to that and class I RNase T2 protein from and to a RNS2, can and in analyses also indicated that mutants to and that are after et al., 2019). These that specific and be by different be associated with different biological roles and of although both and with proteins in plants et al., 2017; et al., 2017). is the that a large number of in the cytoplasm and can with although the RNases involved in their processing are not et al., 2017). Similarly, least two have been proposed for the of in et a large number of in and that of a from is and showed that purified can this tRNA in On the other hand, et in that of the activity of a of the RNase A family, and of this enzyme of the in the of is also for the production of in and several RNase T2 enzymes to the production of in the and and purified can in et al., that RNase T2 enzymes have a conserved role in production in a role for plant RNase T2 proteins in the turnover of and evidence that also in the production of the enzymes and their are not found in the and in the but is a vacuolar enzyme et al., 1999; et al., et al., 2017) and likely to is mainly of the et al., 1999). As in the of to the vacuole can be by RNS2, but the tRNA processing to is are to an or the RNases are the cytoplasm to on is not known. the were the that of RNases with specificity the cytoplasm should also be (see class I RNases are also associated with responses to abiotic and biotic (Table 1). In is to a related to a biological for in defense RNA or phosphate in other their and the specific role in stress responses is less It is to that the role of RNase T2 enzymes in stress responses also be to their to generate thus a role in these and have a role in the functional are for RNA with and 3′ processing and RNA decay and et al., Although RNases were by the Arabidopsis genome for RNase and this to be Some RNases were shown to carry other functions for the et al., 2014), and RNases in of with a for the et al., 2018). as evidence of all RNase are present in bacteria and et al., et al., 2018). on their or and the associated in RNA there are RNases roles are the 5′-3′ the whereas two 3′-5′ exoribonucleases, and the 3′ is by but the of and is et al., for a of RNA These RNases are not specific and on a variety of The roles of ribonucleases in and mRNA In RNase the end of whereas and the 3′ The is also to by several but the of RNase RNase and is of the final are from degradation by RNA proteins and or are the 3′ end by the of a RNase also in RNA degradation and role the of RNA is not In endoribonucleases have a role in through their with proteins that RNA by or endoribonucleases of the and can or two genes and or and 3′ As in and the 3′ 3′ are from degradation by or are the 3′ end by the of a the 3′-5′ to the of however, the of this activity is RNase protein of RNase RNA by et of of the two exoribonucleases, and RNase to in 3′ end for both mRNAs and and of both in Arabidopsis et al., et al., 2012). the first enzyme to RNA in et al., role however, to degrade RNA through a 3′-5′ and the the two activities on the of and the of whereas degradation et al., In the activity is for the of to but their biological role is et al., 2011). from the the active purified from is a of three that not seem to with other ribonucleases et al., 2001; et al., in research has shown that is necessary to RNA 3′ and that activity is by or et al., et al., 2001; et al., et al., 2011). has a for a in RNA decay in et al., et al., and Arabidopsis mutants have and are but can to et al., 2009; et al., 2011). 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It has been proposed that the activity be in active ribonucleases and this the in and in of RNase can be found in most plants and with the exception of et al., It is an essential in involved in both mRNA and degradation and In RNA that the plant enzyme is an with a for single-stranded RNA with and a in with the of the bacterial enzyme et al., et al., RNase is found in complexes in the of the in an et al., mutants with are and the essential role of RNase in plants et al., et al., 2010). the of that the enzyme be involved in of the mRNA et al., 2010; et al., 2012). It proposed that the for mutants the of the to express of the ribosomal proteins et al., 2010). 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Similarly, of the protein to the a by of the site et al., RNA homeostasis is essential to maintain cellular function. In to the that in cytoplasmic and nuclear RNA is that organellar and secretory RNases have an important role in this RNases in and maintain a functional not only mRNA but also of and other noncoding RNA essential for their function. Similarly, secretory RNases RNA to primary and have also been for a variety of stress response their we do not have a of their activities and the number of RNases that are in in we do not the in of these RNases and the are to the RNase or RNases to carry out their specific functions. although is that many of these RNases have to their activity to the RNA in a or pathway, on of organellar and secretory RNases, and that control their activity (see This is by the of the RNA control in the ribonucleases in of processing also function in RNA do to or degrade In to the degradation and 2009; et al., and the of and RNA proteins et al., et al., et al., three have been biochemical evidence showed that RNases do not and are of with that their functions et al., et al., 2012). It is to that of these a role in RNA proteins also a RNase to a specific RNA to has been in mitochondria. have shown that RNases can be for example through As proposed for this a to the RNase specificity activity RNase activity be through the of both enzymes and to has been in bacteria It for example proposed that the and be on their in the and et al., 2012). These questions should research in the we are to have a of RNA homeostasis in The and functional are of to the in this RNase RNase RNase RNase all the members of and present for

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