RNA-Binding Proteins Required for Chloroplast RNA Processing

Abstract

Chloroplasts and mitochondria both evolved from endosymbionts and therefore share many physiologic, biochemical, and genetic features. This chapter reviews a selection of chloroplast RNA-binding proteins, specifically those from land plants that meet the following criteria: (1) the encoding gene is known; (2) an association with RNA has been demonstrated; and (3) the protein does not have any (known) catalytic activity. This set of RNA-binding proteins predominantly consists of members from five protein families: the pentatricopeptide repeat (PPR) protein family; the chloroplast ribonucleoprotein (cpRNP) family; the chloroplast ribosome maturation (CRM) and RNA splicing protein family; the Whirly (Why) protein family; and the plant organelle RNA recognition (PORR) protein family. The functions of representative members of these families are summarized, and their protein–RNA interactions are discussed.

Glossary

Arm-repeat:

Helical hairpin repeat protein named after the armadillo protein. The repeat is about 40 amino acids long and repeated in tandem. It was first identified in the Drosophila melanogaster segment polarity gene armadillo.

Co-IP and slot-blot:

A technique related to RIP-Chip. However, instead of using a microarray for detection, co-precipitated RNA is blotted on a membrane using a vacuum manifold. DNA probes (e.g., PCR products) are labeled radioactively and hybridized to the membrane similar to an RNA gel blot hybridization experiment.

EMSA:

The electrophoretic mobility shift assay (EMSA), also referred to as the gel retardation assay or gel shift assay, is a common technique used to characterize protein–RNA interactions (also done with DNA). EMSA takes advantage of the fact that complexes of protein and RNA migrate through a nondenaturing polyacrylamide gel more slowly than free RNA fragments. The assay is carried out by incubating a purified protein with labeled RNA containing the putative protein binding site and nonspecific competitor RNA.

Filter binding assay:

Filter binding assays measure affinities between RNA (or DNA) and protein molecules using a double layer of different filters. The top filter is made of nitrocellulose and binds proteins but not nucleic acids. However, if RNA is sticking to the protein it will be retained on this filter already. The second, bottom filter is a highly positively charged nylon membrane that will bind all nucleic acids that have passed the first filter. The exact amount of RNA sticking to each of the two filters can be quantified if the RNA was radioactively labeled, for example. The ratio of bound to unbound RNA together can be correlated with protein input and competitor RNA in the initial binding reaction. Among others, the data can be used to determine the binding constant for protein–RNA interactions.

HEAT-repeat:

Four proteins with simple, iterated helical-hairpin repeat domains gave rise to this acronym (Huntingtin, elongation factor 3 [EF3], protein phosphatase 2A [PP2A], and PI3-kinase [TOR1]). The domain is often found in proteins involved in transport processes.

Hydroxyradical footprinting:

Hydroxyradical footprinting is a technique to determine the binding site of an RNA binding protein on its RNA target (also done with DNA-binding proteins). The recombinant purified protein is incubated with its cognate target and attacked with hydroxyl radicals. The radicals will destroy single-stranded RNA by attacking the phosphodiester connections between ribonucleotides in single-stranded areas.

KH-domain:

The K homology (KH) domain was first identified in the human heterogeneous nuclear ribonucleoprotein (hnRNP) K. It is a wide-spread RNA binding motif of around 70 amino acids and is often found in multiple copies within one protein.

Puf-repeat:

Named after the human Pumilio protein, the Puf proteins contain a C-terminal domain that consist of repeated helical hairpin repeat domains, the Puf domains. Puf proteins regulate various aspects of development by controlling mRNA stability and translation through sequence-specific interactions, often with sequence elements in the 3′ untranslated region of target mRNAs.

Ribonucleoprotein (RNP):

Ribonucleoprotein (RNP) originally referred to proteins localized to the nucleus and associated with RNA. A more recent definition includes all particles that combine ribonucleic acid and protein together.

RIP-Chip:

RNA-coimmunoprecipitation and chip analysis refers to a technique that couples immunoprecipitation of an RNP with subsequent analysis of the bound RNA using a microarray. This allows detecting targets of an RNA binding protein across an entire genome.

SMR-Domain:

The small MutS-related (SMR) domain has been implicated in recombination and repair processes. It is related to the C-terminal part of the bacterial MutS2 domain and is found in bacteria as well as eukaryotes.

UV-cross-linking:

A technique related to EMSAs, where protein–nucleic acid complexes are irradiated with ultraviolet light. This causes covalent bonds to form between the nucleic acid and proteins that are in close contact with the nucleic acid. The protein–RNA adduct is often treated with RNAses to remove non-cross-linked RNA. For detection, the RNA is often labeled and can be visualized in a denaturing polyacrylamide gel.