A Role for MINIYO and QUATRE-QUART2 in the Assembly of RNA Polymerases II, IV, and V in Arabidopsis

Abstract

RNA polymerases IV and V (Pol IV and Pol V) are required for the generation of noncoding RNAs in RNA-directed DNA methylation (RdDM). Their subunit compositions resemble that of Pol II. The mechanism and accessory factors involved in their assembly remain largely unknown. In this study, we identified mutant alleles of (), (), and () that cause defects in RdDM in We found that Pol IV-dependent small interfering RNAs and Pol V-dependent transcripts were greatly reduced in the mutants. NRPE1, the largest subunit of Pol V, failed to associate with other Pol V subunits in the and mutants, suggesting the involvement of IYO and QQT2 in Pol V assembly. In addition, we found that IYO and QQT2 were mutually dependent for their association with the NRPE3 subassembly prior to the assembly of Pol V holoenzyme. Finally, we show that IYO and QQT2 are similarly required for the assembly of Pol II and Pol IV. Our findings reveal IYO and QQT2 as cofactors for the assembly of Pol II, Pol IV, and Pol V and provide mechanistic insights into how RNA polymerases are assembled in plants.

Innovation, conservation and repurposing of gene function in plant root cell type development

“…35 In concordance with the clustering analysis, most of these genes show specific expression in the meristematic zone. Among these genes is QQT2, which is essential for correct cell divisions during embryogenesis (32) and required for the assembly of RNA polymerases II, IV, and V (33). Additional conserved meristematic zone-enriched genes encode two nucleoporins, SAR3 and NUP43, subunits of the nuclear pore complex, which regulate nucleocytoplasmic transport of 40 protein and RNA and play important roles in hormone signaling and developmental processes (34).…”

Identification of Domains and Factors Involved in MINIYO Nuclear Import

“…Previous findings revealed that IYO interacts with GPN1 and GPN2 (Muñoz et al, 2017; Li et al, 2018), two members of the GPN family of GTPases that have been linked to selective nuclear transport of Pol II (Forget et al, 2010; Carre and Shiekhattar, 2011; Staresincic et al, 2011; Minaker et al, 2013; Zeng et al, 2018). In Arabidopsis , there are three GPNs, GPN1 to GPN3.…”

“…Pol II subunits do not have NLSs, so the Pol II complex associates with the NLS-containing protein IWR1 for import into the yeast nucleus, a function that appears to be also conserved in human IWR1 (Czeko et al, 2011). Interestingly, IYO and GPN1 have been found in a Pol II subassembly complex that includes the Arabidopsis IWR1 homologue DMS4 (Li et al, 2018). It is thus plausible that the NLSs from IYO and DMS4 cooperate for import of a macro-complex formed by Pol II subunits and associated RPAP factors.…”

“…GPNs are a family of GTPases related to the RAN nuclear transport factors that have been linked to selective nuclear transport of Pol II in humans and yeast (Forget et al, 2010; Calera et al, 2011; Carre and Shiekhattar, 2011; Staresincic et al, 2011; Minaker et al, 2013; Zeng et al, 2018). The interaction between IYO/RPAP1 and GPNs has been conserved throughout evolution in yeast, mammals, and plants (Boulon et al, 2010; Muñoz et al, 2017; Li et al, 2018; Zeng et al, 2018) and may occur through common domains. The IYO/RPAP1 homologues in yeasts encode shorter proteins (439 aa in Saccharomyces cerevisiae ) that correspond essentially to the N-terminal end of plant and mammalian IYO/RPAP1 proteins, including the conserved RPAP1_N and RPA1_C domains.…”

“…In yeast and animal cells, RPAPs constitute a network of cross-interacting factors that participate in the cytosolic assembly of the RNA polymerase II (Pol II) macro-complex and in its nuclear import (Jeronimo et al, 2007; Gibney et al, 2008; Forget et al, 2010; Forget et al, 2013). In Arabidopsis , IYO associates with the RPAP2 orthologue RIMA and the RPAP4 orthologue GPN1 (Muñoz et al, 2017; Li et al, 2018), indicating that the RPAP interaction network is conserved in plants. Interestingly, disrupting RIMA function leads to an increase in the cytosolic/nuclear ratio of IYO, while IYO overexpression increases RIMA nuclear levels (Muñoz et al, 2017), suggesting that these plant RPAPs cooperate for import into the nucleus.…”