The chloroplastic DEVH‐box RNA helicase INCREASED SIZE EXCLUSION LIMIT 2 involved in plasmodesmata regulation is required for group II intron splicing

Abstract

INCREASED SIZE EXCLUSION LIMIT 2 (ISE2) encodes a putative DEVH-box RNA helicase originally identified through a genetic screening for Arabidopsis mutants altered in plasmodesmata (PD) aperture. Depletion of ISE2 also affects chloroplasts activity, decreases accumulation of photosynthetic pigments and alters expression of photosynthetic genes. In this work, we show the chloroplast localization of ISE2 and decipher its role in plastidic RNA processing and, consequently, PD function. Group II intron-containing RNAs from chloroplasts exhibit defective splicing in ise2 mutants and ISE2-silenced plants, compromising plastid viability. Furthermore, RNA immunoprecipitation suggests that ISE2 binds in vivo to several splicing-regulated RNAs. Finally, we show that the chloroplast clpr2 mutant (defective in a subunit of a plastidic Clp protease) also exhibits abnormal PD function during embryogenesis, supporting the idea that chloroplast RNA processing is required to regulate cell-cell communication in plants.

Ties that bind: the integration of plastid signalling pathways in plant cell metabolism

“…ISE2–GFP fusion proteins localize to punctae within the plastid stroma, which is consistent with its previous identification in plastid nucleoids [63]. It is now clear that ISE2 has multiple roles in plastid RNA processing (Figure 3): (i) ISE2 promotes splicing of some, but not all, group II introns [64,65], (ii) ISE2 is required for C-to-U post-transcriptional RNA editing, (iii) ISE2 controls the steady-state levels of dozens of plastid transcripts, broadly promoting accumulation of transcripts encoding pETC components but repressing accumulation of several transcripts encoding ribosomal proteins and RNA polymerase, (iv) ISE2 is similarly required for accumulation of plastid-encoded pETC proteins, and (v) ISE2 is necessary for ribosomal RNA processing and accumulation [65]. In RNA immunoprecipitation experiments, ISE2 was shown to interact with over half of plastid RNA species, including nearly all of the transcripts that require ISE2 for proper splicing and C-to-U editing, suggesting that ISE2 acts directly on a number of RNA species (in concert with other proteins) to regulate plastid RNA metabolism.…”

The Nuclear Ribonucleoprotein SmD1 Interplays with Splicing, RNA Quality Control, and Posttranscriptional Gene Silencing in Arabidopsis

“…Briefly, fixed material was ground in liquid nitrogen and homogenized and nuclei isolated and lysed according to Gendrel et al (2005).RNA immunoprecipitation was basically performed as described by Carlotto et al (2016). The nuclei extract (input) was used for the immunoprecipitation performed by the Direct ChIP Protocol of the Diagenode IP-Star SX-86 Compact robot, using 50 mL of Dynabeads-Protein A (Novex 10008D; Life Technologies) and anti-GFP antibodies (632381; Clontech).…”

The essential chloroplast ribosomal protein uL15c interacts with the chloroplast RNA helicase ISE2 and affects intercellular trafficking through plasmodesmata

“…The data presented here support the hypothesis that the functional status of chloroplasts can influence intercellular trafficking flux via PD. Also lending support to this hypothesis is the finding that the clpr2 mutant has increased intercellular trafficking (Carlotto et al ., ).…”