THO2, a core member of the THO/TREX complex, is required for microRNA production in Arabidopsis

Abstract

SUMMARYThe THO/TREX complex mediates transport of nascent mRNAs from the nucleus towards the cytoplasm in animals, and has a role in small interfering RNA-dependent processes in plants. Here we describe five mutant alleles of Arabidopsis thaliana THO2, which encodes a core subunit of the plant THO/TREX complex. tho2 mutants present strong developmental defects resembling those in plants compromised in microRNA (miRNA) activity. In agreement, not only were the levels of siRNAs reduced in tho2 mutants, but also those of mature miRNAs. As a consequence, a feedback mechanism is triggered, increasing the amount of miRNA precursors, and finally causing accumulation of miRNA-targeted mRNAs. Yeast two-hybrid experiments and confocal microscopy showed that THO2 does not appear to interact with any of the known miRNA biogenesis components, but rather with the splicing machinery, implying an indirect role of THO2 in small RNA biogenesis. Using an RNA immunoprecipitation approach, we found that THO2 interacts with miRNA precursors, and that tho2 mutants fail to recruit such precursors into the miRNA-processing complex, explaining the reduction in miRNA production in this mutant background. We also detected alterations in the splicing pattern of genes encoding serine/arginine-rich proteins in tho2 mutants, supporting a previously unappreciated role of the THO/TREX complex in alternative splicing.

The ‘how’ and ‘where’ of plant microRNAs

“…Proteins belonging to this group include CAP-BINDING PROTEIN 80 (CBP80) and CAP-BINDING PROTEIN 20 (CBP20) (Laubinger et al ., 2008; reviewed in Gonatopoulos-Pournatzis & Cowling, 2014), STABILIZED1 (STA1) (Ben Chaabane et al ., 2013), THO1/HPR1/EMU and THO2 (Furumizu et al ., 2010; Francisco-Mangilet et al ., 2015), SICKLE (Zhan et al ., 2012), TOUGH (Ren et al ., 2012b), PSR1-INTERACTING PROTEIN 1 (PINP1) (Qiao et al ., 2015) and MODIFIER OF SNC1, 2 (MOS2) (X. Wu et al ., 2013).…”

“…AtGRP7 overexpression results in changes in pri-miRNA splicing (Streitner et al ., 2012; Koster et al ., 2014) MOS2 is required for appropriate splicing of SNC1 ( SUPPRESSOR OF NPR1-1, CONSTITUTIVE 1 ), which encodes a Toll Interleukin 1 Receptor Nucleotide Binding Leucine-Rich Repeat (TIR-NB-LRR) class of protein involved in plant defense responses (Zhang et al ., 2005; Copeland et al ., 2013). Alternatively spliced SR gene transcripts were detected in tho1 and tho2 mutants (Furumizu et al ., 2010; Francisco-Mangilet et al ., 2015). PRL1 belongs to the NineTeen Complex (NTC) or MOS4-associated complex (MAC), a protein complex with 19 conserved members in yeast, human and plants.…”

“…For example, the levels of pri-miR159a, which contains no introns, were altered in the mutants of CBC, STA1, PRL1, MOS2, THO2 and SICKLE (Laubinger et al ., 2008; Szarzynska et al ., 2009; Zhan et al ., 2012; Ben Chaabane et al ., 2013; Copeland et al ., 2013; X. Wu et al ., 2013; Zhang et al ., 2014; Francisco-Mangilet et al ., 2015). At least for pri-miRNAs without introns, the aforementioned proteins cannot act in miRNA biogenesis through their functions in RNA splicing.…”

The SnRK2 kinases modulate miRNA accumulation in Arabidopsis

“…STABLIZED 1 (STA1) and XAP5 CIRCADIAN TIMEKEEPER (XCT) can modulate miRNA accumulation through regulation of DCL1 transcript level [22, 23]. Several other proteins, such as SICKLE (SIC), Modifier of SNC1, 2 (MOS2), AtGRP7 and THO2 are additional players involved in miRNA accumulation [24–27]. miRNAs in both animals and plants are processed from primary miRNA (pri-miRNA) transcripts, which include imperfect stem-loop structures.…”

KH domain protein RCF3 is a tissue-biased regulator of the plant miRNA biogenesis cofactor HYL1

“…In plants, after transcription of an MIRNA gene by RNA polymerase II, the primary miRNA (pri-miRNA) is incorporated into nuclear bodies known as D-bodies (1). There, it undergoes a two-step maturation process orchestrated by the ribonuclease DICER-LIKE1 (DCL1) (2).…”