A protein-RNA complex orchestrated by EMB1006, EMB1270, EMB976, and CFM2 facilitates clpP1 intron 2 splicing in Arabidopsis chloroplasts

Abstract

In Arabidopsis, the PPR proteins EMB1006, EMB1270, and EMB976 are all essential for the splicing of plastid clpP1 intron 2 (clpP1.2), although each also targets other distinct RNAs. The precise mechanism underlying their co-ordinated action in clpP1.2 splicing remains unclear. In this study, RNA electrophoretic mobility shift assays, guided by PPR code prediction, confirm that EMB1006 specifically binds to a sequence near the 3′ end of clpP1 exon 2. Additionally, immunoprecipitation coupled with mass spectrometry reveals that EMB1006 forms a complex with EMB1270, EMB976, and CFM2. Direct interactions between EMB1006 and EMB1270 or CFM2 are further supported by yeast two-hybrid (Y2H) and semi-in vivo pull-down assays. However, no direct interactions between EMB976 and EMB1006, CFM2 or EMB1270 are detected by Y2H. Based on these findings and previous evidence that EMB1270 binds to clpP1 intron 2 and interacts with CFM2, we propose a model in which EMB1006 and EMB1270 bind to distinct sites on clpP1 pre-mRNA. Together with CFM2 and possible indirect association with EMB976, they assemble into a protein-RNA complex that facilitates the splicing of clpP1.2 in chloroplasts.

1. Introduction

Plastids and mitochondria are semi-autonomous organelles whose gene expression is tightly regulated by a multitude of nucleus-encoded proteins [1]. Among these regulators, pentatricopeptide repeat (PPR) proteins constitute the largest family involved in post-transcriptional regulation within the organelles of higher plants. These proteins are characterized by tandem repeats of an ~35-amino-acid motif, with each protein containing 2–30 such repeats. Based on the features of these PPR motifs, the family is divided into P and PLS subfamilies [2]. A key characteristic of PPR proteins is their ability to bind to specific RNA sequences through a “one-repeat-to-one-nucleotide” recognition mechanism. It is well established that they play diverse roles in organellar RNA processing. Generally, P-type PPRs are implicated in RNA stabilization, cleavage, and intron splicing, whereas PLS-type members primarily participate in RNA editing [3].

In the Arabidopsis plastid genome, the twenty introns are categorized into group I (represented by a single intron) and group II (comprising 19 introns) based on their splicing mechanisms [4,5]. Since group II introns have entirely lost the capacity for self-splicing, their removal depends on numerous nucleus-encoded factors. Beyond PPR proteins, other RNA-binding proteins are also crucial for group II intron splicing. These include chloroplast RNA splicing 1 (CRS1), CRS2-associated factors (CAF1 and CAF2) [6], and chloroplast RNA splicing and ribosome maturation (CRM) family members (CFM1, CFM2, and CFM3) [7–9]. Notably, many of these proteins, such as CRS1, CAF1, CAF2, CFM1, CFM2, and CFM3, share a conserved CRM domain. Furthermore, RNC1, a ribonuclease III domain protein initially identified in maize chloroplasts as part of CAF1/CAF2 complexes, is required for the splicing of several group II introns [10]. Another key factor is WTF1 (What’s This Factor 1), which contains an RNA-binding DUF860 domain and interacts with RNC1 to promote the splicing of multiple group II introns [11]. Given that mutations in RNC1 and WTF1 disrupt the splicing of most plastid introns, they are considered general splicing factors. In contrast, PPR proteins are hypothesized to act earlier by conferring sequence specificity. They are thought to initiate splicing by binding to specific RNA sequences and altering the RNA structure, thereby facilitating the recruitment of general splicing complexes such as those containing WTF1/RNC1 or CRM proteins [1]. This functional model is supported by recent studies in both mitochondria and chloroplasts. For instance, in Arabidopsis chloroplasts, the PPR protein EMB1270 interacts with the CRM-domain protein CFM2 to promote the splicing of specific group II introns [5]. Similarly, in maize mitochondria, the PPR protein PPR14, which functions through physical interactions with PPR-SMR1 and the CRM protein Zm-mCSF1, is essential for the splicing of mitochondrial nad2 intron 3 and nad7 introns 1 and 2 [12]. Furthermore, Small PPR Protein 2 (SPR2), which contains only four PPR repeats, participates in the splicing of over half of the introns in maize mitochondria by interacting with the small MutS-related domain protein PPR-SMR1. Both SPR2 and PPR-SMR1 can also interact with additional splicing factors, including the PPR proteins EMPTY PERICARP16 and PPR14, as well as the CRM protein Zm-mCSF1, which are involved in the splicing of specific introns [13].

The caseinolytic protease (Clp) is essential for plastid development and function, primarily by degrading misfolded, aggregated, or otherwise unwanted proteins [14]. The Clp system comprises a tetradecameric proteolytic core—composed of catalytic...