Peroxisomes house critical metabolic reactions that are essential for seedling development. As seedlings mature, metabolic requirements change, and peroxisomal contents are remodeled. The resident peroxisomal protease LON2 is positioned to degrade obsolete or damaged peroxisomal proteins, but data supporting such a role in plants have remained elusive. Arabidopsis thaliana lon2 mutants display defects in peroxisomal metabolism and matrix protein import but appear to degrade matrix proteins normally. To elucidate LON2 functions, we executed a forward-genetic screen for lon2 suppressors, which revealed multiple mutations in key autophagy genes. Disabling core autophagy-related gene (ATG) products prevents autophagy, a process through which cytosolic constituents, including organelles, can be targeted for vacuolar degradation. We found that atg2, atg3, and atg7 mutations suppressed lon2 defects in auxin metabolism and matrix protein processing and rescued the abnormally large size and small number of lon2 peroxisomes. Moreover, analysis of lon2 atg mutants uncovered an apparent role for LON2 in matrix protein turnover. Our data suggest that LON2 facilitates matrix protein degradation during peroxisome content remodeling, provide evidence for the existence of pexophagy in plants, and indicate that peroxisome destruction via autophagy is enhanced when LON2 is absent.

Summary The CCHC-type Zinc Finger Nucleic Acid Binding Protein (CNBP/ZNF9) is conserved in eukaryotes and essential for embryonic development in mammals. It has been implicated in transcriptional as well as post-transcriptional gene regulation; however, its nucleic acid ligands and molecular function remain elusive. Here, we use multiple systems-wide approaches to identify CNBP targets and function. We used Photoactivatable Ribonucleoside Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) to identify 8420 CNBP binding sites on 4178 mRNAs. CNBP preferentially bound G-rich elements in the target mRNA coding sequences, most of which were previously found to form G-quadruplex and other stable structures in vitro. Functional analyses, including RNA sequencing, ribosome profiling, and quantitative mass spectrometry, revealed that CNBP binding did not influence target mRNA abundance but rather increased their translational efficiency. Considering that CNBP binding prevented G quadruplex structure formation in vitro, we hypothesize that CNBP is supporting translation by resolving stable structures on mRNAs.

During post-transcriptional gene regulation (PTGR), RNA binding proteins (RBPs) interact with all classes of RNA to control RNA maturation, stability, transport, and translation. Here, we describe Photoactivatable-Ribonucleoside-Enhanced Cross linking and Immunoprecipitation (PAR-CLIP), a transcriptome-scale method for identifying RBP binding sites on target RNAs with nucleotide-level resolution. This method is readily applicable to any protein directly contacting RNA, including RBPs that are predicted to bind in a sequence- or structure-dependent manner at discrete RNA recognition elements (RREs), and those that are thought to bind transiently, such as RNA polymerases or helicases.

Disrupting autophagy restores peroxisome function to an Arabidopsis lon2 mutant and reveals a role for the LON2 protease in peroxisomal matrix protein degradation.