Kenneth P. Watkins

Nucleoid-enriched proteomes in developing plastids and chloroplasts from maize leaves; a new conceptual framework for nucleoid functions

Plastids contain multiple copies of the plastid chromosome, folded together with proteins and RNA into nucleoids. The degree to which components of the plastid gene expression and protein biogenesis machineries are nucleoid associated, and the factors involved in plastid DNA organization, repair, and replication, are poorly understood. To provide a conceptual framework for nucleoid function, we characterized the proteomes of highly enriched nucleoid fractions of proplastids and mature chloroplasts isolated from the maize (Zea mays) leaf base and tip, respectively, using mass spectrometry. Quantitative comparisons with proteomes of unfractionated proplastids and chloroplasts facilitated the determination of nucleoid-enriched proteins. This nucleoid-enriched proteome included proteins involved in DNA replication, organization, and repair as well as transcription, mRNA processing, splicing, and editing. Many proteins of unknown function, including pentatricopeptide repeat (PPR), tetratricopeptide repeat (TPR), DnaJ, and mitochondrial transcription factor (mTERF) domain proteins, were identified. Strikingly, 70S ribosome and ribosome assembly factors were strongly overrepresented in nucleoid fractions, but protein chaperones were not. Our analysis strongly suggests that mRNA processing, splicing, and editing, as well as ribosome assembly, take place in association with the nucleoid, suggesting that these processes occur cotranscriptionally. The plastid developmental state did not dramatically change the nucleoid-enriched proteome but did quantitatively shift the predominating function from RNA metabolism in undeveloped plastids to translation and homeostasis in chloroplasts. This study extends the known maize plastid proteome by hundreds of proteins, including more than 40 PPR and mTERF domain proteins, and provides a resource for targeted studies on plastid gene expression. Details of protein identification and annotation are provided in the Plant Proteome Database.

A member of the Whirly family is a multifunctional RNA- and DNA-binding protein that is essential for chloroplast biogenesis

‘Whirly’ proteins comprise a plant-specific protein family whose members have been described as DNA-binding proteins that influence nuclear transcription and telomere maintenance, and that associate with nucleoids in chloroplasts and mitochondria. We identified the maize WHY1 ortholog among proteins that coimmunoprecipitate with CRS1, which promotes the splicing of the chloroplast atpF group II intron. ZmWHY1 localizes to the chloroplast stroma and to the thylakoid membrane, to which it is tethered by DNA. Genome-wide coimmunoprecipitation assays showed that ZmWHY1 in chloroplast extract is associated with DNA from throughout the plastid genome and with a subset of plastid RNAs that includes atpF transcripts. Furthermore, ZmWHY1 binds both RNA and DNA in vitro. A severe ZmWhy1 mutant allele conditions albino seedlings lacking plastid ribosomes; these exhibit the altered plastid RNA profile characteristic of ribosome-less plastids. Hypomorphic ZmWhy1 mutants exhibit reduced atpF intron splicing and a reduced content of plastid ribosomes; aberrant 23S rRNA metabolism in these mutants suggests that a defect in the biogenesis of the large ribosomal subunit underlies the ribosome deficiency. However, these mutants contain near normal levels of chloroplast DNA and RNAs, suggesting that ZmWHY1 is not directly required for either DNA replication or for global plastid transcription.

A Ribonuclease III Domain Protein Functions in Group II Intron Splicing in Maize Chloroplasts

Chloroplast genomes in land plants harbor ∼20 group II introns. Genetic approaches have identified proteins involved in the splicing of many of these introns, but the proteins identified to date cannot account for the large size of intron ribonucleoprotein complexes and are not sufficient to reconstitute splicing in vitro. Here, we describe an additional protein that promotes chloroplast group II intron splicing in vivo. This protein, RNC1, was identified by mass spectrometry analysis of maize (Zea mays) proteins that coimmunoprecipitate with two previously identified chloroplast splicing factors, CAF1 and CAF2. RNC1 is a plant-specific protein that contains two ribonuclease III (RNase III) domains, the domain that harbors the active site of RNase III and Dicer enzymes. However, several amino acids that are essential for catalysis by RNase III and Dicer are missing from the RNase III domains in RNC1. RNC1 is found in complexes with a subset of chloroplast group II introns that includes but is not limited to CAF1- and CAF2-dependent introns. The splicing of many of the introns with which it associates is disrupted in maize rnc1 insertion mutants, indicating that RNC1 facilitates splicing in vivo. Recombinant RNC1 binds both single-stranded and double-stranded RNA with no discernible sequence specificity and lacks endonuclease activity. These results suggest that RNC1 is recruited to specific introns via protein–protein interactions and that its role in splicing involves RNA binding but not RNA cleavage activity.

A plant-specific RNA-binding domain revealed through analysis of chloroplast group II intron splicing

Comparative genomics has provided evidence for numerous conserved protein domains whose functions remain unknown. We identified a protein harboring “domain of unknown function 860” (DUF860) as a component of group II intron ribonucleoprotein particles in maize chloroplasts. This protein, assigned the name WTF1 (“whats this factor?”), coimmunoprecipitates from chloroplast extract with group II intron RNAs, is required for the splicing of the introns with which it associates, and promotes splicing in the context of a heterodimer with the RNase III-domain protein RNC1. Both WTF1 and its resident DUF860 bind RNA in vitro, demonstrating that DUF860 is a previously unrecognized RNA-binding domain. DUF860 is found only in plants, where it is represented in a protein family comprising 14 orthologous groups in angiosperms. Most members of the DUF860 family are predicted to localize to chloroplasts or mitochondria, suggesting that proteins with this domain have multiple roles in RNA metabolism in both organelles. These findings add to emerging evidence that the coevolution of nuclear and organellar genomes spurred the evolution of diverse noncanonical RNA-binding motifs that perform organelle-specific functions.

CRS1, a Chloroplast Group II Intron Splicing Factor, Promotes Intron Folding through Specific Interactions with Two Intron Domains

Group II introns are ribozymes that catalyze a splicing reaction with the same chemical steps as spliceosome-mediated splicing. Many group II introns have lost the capacity to self-splice while acquiring compensatory interactions with host-derived protein cofactors. Degenerate group II introns are particularly abundant in the organellar genomes of plants, where their requirement for nuclear-encoded splicing factors provides a means for the integration of nuclear and organellar functions. We present a biochemical analysis of the interactions between a nuclear-encoded group II splicing factor and its chloroplast intron target. The maize (Zea mays) protein Chloroplast RNA Splicing 1 (CRS1) is required specifically for the splicing of the group II intron in the chloroplast atpF gene and belongs to a plant-specific protein family defined by a recently recognized RNA binding domain, the CRM domain. We show that CRS1s specificity for the atpF intron in vivo can be explained by CRS1s intrinsic RNA binding properties. CRS1 binds in vitro with high affinity and specificity to atpF intron RNA and does so through the recognition of elements in intron domains I and IV. These binding sites are not conserved in other group II introns, accounting for CRS1s intron specificity. In the absence of CRS1, the atpF intron has little uniform tertiary structure even at elevated [Mg2+]. CRS1 binding reorganizes the RNA, such that intron elements expected to be at the catalytic core become less accessible to solvent. We conclude that CRS1 promotes the folding of its group II intron target through tight and specific interactions with two peripheral intron segments.

A Rapid Ribosome Profiling Method Elucidates Chloroplast Ribosome Behavior in Vivo

Ribosome profiling provides a quantitative readout of mRNA segments bound by ribosomes in vivo. This work describes a cost-effective variant of the method and use it to address unresolved questions in maize plastid gene expression, including the translational coupling of overlapping reading frames, the ability of unspliced chloroplast RNAs to engage in translation, and mechanisms of ribosome pausing. The profiling of ribosome footprints by deep sequencing has revolutionized the analysis of translation by mapping ribosomes with high resolution on a genome-wide scale. We present a variation on this approach that offers a rapid and cost-effective alternative for the genome-wide profiling of chloroplast ribosomes. Ribosome footprints from leaf tissue are hybridized to oligonucleotide tiling microarrays of the plastid ORFeome and report the abundance and translational status of every chloroplast mRNA. Each assay replaces several time-consuming traditional methods while also providing information that was previously inaccessible. To illustrate the utility of the approach, we show that it detects known defects in chloroplast gene expression in several nuclear mutants of maize (Zea mays) and that it reveals previously unsuspected defects. Furthermore, it provided firm answers to several lingering questions in chloroplast gene expression: (1) the overlapping atpB/atpE open reading frames, whose translation had been proposed to be coupled, are translated independently in vivo; (2) splicing is not a prerequisite for translation initiation on an intron-containing chloroplast RNA; and (3) a feedback control mechanism that links the synthesis of ATP synthase subunits in Chlamydomonas reinhardtii does not exist in maize. An analogous approach is likely to be useful for studies of mitochondrial gene expression.

Chloroplast RH3 DEAD Box RNA Helicases in Maize and Arabidopsis Function in Splicing of Specific Group II Introns and Affect Chloroplast Ribosome Biogenesis

Chloroplasts in angiosperms contain at least seven nucleus-encoded members of the DEAD box RNA helicase family. Phylogenetic analysis shows that five of these plastid members (RH22, -39, -47, -50, and -58) form a single clade and that RH3 forms a clade with two mitochondrial RH proteins (PMH1 and -2) functioning in intron splicing. The function of chloroplast RH3 in maize (Zea mays; ZmRH3) and Arabidopsis (Arabidopsis thaliana; AtRH3) was determined. ZmRH3 and AtRH3 are both under strong developmental control, and ZmRH3 abundance sharply peaked in the sink-source transition zone of developing maize leaves, coincident with the plastid biogenesis machinery. ZmRH3 coimmunoprecipitated with a specific set of plastid RNAs, including several group II introns, as well as pre23S and 23S ribosomal RNA (rRNA), but not 16S rRNA. Furthermore, ZmRH3 associated with 50S preribosome particles as well as nucleoids. AtRH3 null mutants are embryo lethal, whereas a weak allele (rh3-4) results in pale-green seedlings with defects in splicing of several group II introns and rRNA maturation as well as reduced levels of assembled ribosomes. These results provide strong evidence that RH3 functions in the splicing of group II introns and possibly also contributes to the assembly of the 50S ribosomal particle. Previously, we observed 5- to 10-fold up-regulation of AtRH3 in plastid Caseinolytic protease mutants. The results shown here indicate that AtRH3 up-regulation was not a direct consequence of reduced proteolysis but constituted a compensatory response at both RH3 transcript and protein levels to impaired chloroplast biogenesis; this response demonstrates that cross talk between the chloroplast and the nucleus is used to regulate RH3 levels.

APO1 promotes the splicing of chloroplast group II introns and harbors a plant-specific zinc-dependent RNA binding domain.

This study demonstrates that APO1, originally proposed to be involved in the maturation of proteins with [4Fe-4S] ligands, is instead required for the splicing of several chloroplast introns. It shows further that DUF794, the plant-specific domain of unknown function that makes up the bulk of APO1, is an RNA binding domain harboring zinc binding motifs. Arabidopsis thaliana APO1 is required for the accumulation of the chloroplast photosystem I and NADH dehydrogenase complexes and had been proposed to facilitate the incorporation of [4Fe-4S] clusters into these complexes. The identification of maize (Zea mays) APO1 in coimmunoprecipitates with a protein involved in chloroplast RNA splicing prompted us to investigate a role for APO1 in splicing. We show here that APO1 promotes the splicing of several chloroplast group II introns: in Arabidopsis apo1 mutants, ycf3-intron 2 remains completely unspliced, petD intron splicing is strongly reduced, and the splicing of several other introns is compromised. These splicing defects can account for the loss of photosynthetic complexes in apo1 mutants. Recombinant APO1 from both maize and Arabidopsis binds RNA with high affinity in vitro, demonstrating that DUF794, the domain of unknown function that makes up almost the entirety of APO1, is an RNA binding domain. We provide evidence that DUF794 harbors two motifs that resemble zinc fingers, that these bind zinc, and that they are essential for APO1 function. DUF794 is found in a plant-specific protein family whose members are all predicted to localize to mitochondria or chloroplasts. Thus, DUF794 adds a new example to the repertoire of plant-specific RNA binding domains that emerged as a product of nuclear-organellar coevolution.

Ribosome Profiling in Maize

Ribosome profiling (also known as Ribo-seq) provides a genome-wide, high-resolution, and quantitative accounting of mRNA segments that are occupied by ribosomes in vivo. The method has been used to address numerous questions in bacteria, yeast, and metazoa, but its application to questions in plant biology is just beginning. This chapter provides a detailed protocol for profiling ribosomes in plant leaf tissue. The method was developed and optimized with maize, but it has been used successfully with Arabidopsis and tobacco as well. The method captures ribosome footprints from the chloroplast and cytosol in the same preparation, but it is not optimal for detecting the footprints of mitochondrial ribosomes. The protocol is robust and simpler than many of the methods reported previously for ribosome profiling in plants.