ribonucleoprotein capture by in vivo expression of a ...1 1 breakthrough report 2 3...

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BREAKTHROUGH REPORT 1 2

Ribonucleoprotein Capture by in vivo Expression of a Designer 3

Pentatricopeptide Repeat Protein in Arabidopsis 4

James J. McDermott, Kenneth P. Watkins, Rosalind Williams-Carrier, Alice Barkan* 5

Institute of Molecular Biology, University of Oregon, Eugene OR 974056

* Corresponding author. Alice Barkan, Institute of Molecular Biology, University of Oregon,7 Eugene, OR 97405. Email: [email protected]. Tel: 541-346-51458

9 Short Title: Designer PPR protein as RNA affinity tag 10

11 One sentence summary: Artificial proteins built from consensus PPR motifs bind the intended 12 RNA in vivo and can be used as RNA affinity tags to purify endogenous RNPs and identify the 13 bound proteins. 14

15 The author responsible for distribution of materials integral to the findings presented in this 16 article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) 17 is: Alice Barkan ([email protected]). 18

19 ABSTRACT 20 Pentatricopeptide repeat (PPR) proteins bind RNA via a mechanism that facilitates the 21 customization of sequence specificity. However, natural PPR proteins have irregular features 22 that limit the degree to which their specificity can be predicted and customized. We demonstrate 23 here that artificial PPR proteins built from consensus PPR motifs selectively bind the intended 24 RNA in vivo, and we use this property to develop a new tool for ribonucleoprotein 25 characterization. We show by RNA coimmunoprecipitation-sequencing (RIP-seq) that artificial 26 PPR proteins designed to bind the Arabidopsis thaliana chloroplast psbA mRNA bind with high 27 specificity to psbA mRNA in vivo. Analysis of coimmunoprecipitating proteins by mass 28 spectrometry showed the psbA translational activator HCF173 and two RNA-binding proteins of 29 unknown function (CP33C and SRRP1) to be highly enriched. RIP-seq revealed that these 30 proteins are bound primarily to psbA RNA in vivo, and precise mapping of the HCF173 and 31 CP33C binding sites placed them in different locations on psbA mRNA. These results 32 demonstrate that artificial PPR proteins can be tailored to bind specific endogenous RNAs in 33 vivo, add to the toolkit for characterizing native ribonucleoproteins, and open the door to other 34 applications that rely on the ability to target a protein to a specified RNA sequence. 35

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Plant Cell Advance Publication. Published on May 22, 2019, doi:10.1105/tpc.19.00177

©2019 American Society of Plant Biologists. All Rights Reserved

mailto:[email protected]

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INTRODUCTION 39

The ability to target proteins to specified RNA sequences provides an entrée to diverse 40

approaches for manipulating and analyzing RNA-mediated functions. However, the sequence 41

specificities of most RNA binding proteins are difficult to predict because most RNA-binding 42

domains bind short, degenerate sequence motifs and employ variable binding modes (reviewed 43

in Helder et al., 2016). In this context, the Pumilio/FBF (PUF) and pentatricopeptide repeat 44

(PPR) protein families have attracted interest due to their unusual mode of RNA recognition 45

(Chen and Varani, 2013; Yagi et al., 2014; Hall, 2016). PUF and PPR proteins have tandem 46

helical repeating units that bind consecutive nucleotides with a specificity that is largely 47

determined by the identities of amino acids at two positions. These amino acid “codes” have 48

been used to reprogram native proteins to bind new RNA sequences and for the design of 49

artificial proteins with particular sequence specificities (Barkan et al., 2012; Campbell et al., 50

2014; Coquille et al., 2014; Kindgren et al., 2015; Shen et al., 2015; Shen et al., 2016; Colas 51

des Francs-Small et al., 2018; Miranda et al., 2018; Zhao et al., 2018; Bhat et al., 2019; Yan et 52

al., 2019). 53

PUF and PPR proteins also differ in important respects. They bind RNA with opposite 54

polarity, and they employ distinct amino acid combinations to specify each nucleotide (reviewed 55

in Hall, 2016). PUF proteins comprise a small protein family whose members invariably contain 56

eight repeat motifs (Goldstrohm et al., 2018), whereas the PPR family includes over 400 57

members in plants, and the number of PPR motifs per protein ranges from two to approximately 58

thirty (Lurin et al., 2004). PUF proteins generally localize to the cytoplasm and repress the 59

translation or stability of mRNA ligands (reviewed in Wang et al., 2018), while PPR proteins 60

localize almost exclusively to mitochondria and chloroplasts, where they function in RNA 61

stabilization, translational activation, group II intron splicing, RNA cleavage, and RNA editing 62

(reviewed in Barkan and Small, 2014). The evolutionary malleability of PPR architecture and 63

function suggests that the PPR scaffold may be particularly amenable to tailoring RNA binding 64

affinity, kinetics, and sequence specificity for particular applications. 65

The PPR code has been used to recode several natural PPR proteins to bind non-native 66

RNA ligands in vitro (Barkan et al., 2012) and in vivo (Kindgren et al., 2015; Colas des Francs-67

Small et al., 2018; Rojas et al., 2019). However, the engineering of native PPR proteins is 68

complicated by irregularities in their PPR tracts, which result in variable and unpredictable 69

contributions of their PPR motifs to RNA affinity and specificity (Fujii et al., 2013; Okuda et al., 70

2014; Miranda et al., 2017; Rojas et al., 2018). By contrast, artificial PPR proteins (aPPRs) built 71

from consensus PPR motifs exhibit predictable sequence-specificity in vitro (Coquille et al., 72

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2014; Shen et al., 2015; Miranda et al., 2018; Yan et al., 2019). However, the degree to which 73

such proteins bind selectively to RNAs in vivo has not been reported. 74

In this work, we advance efforts to engineer PPR proteins by showing that two aPPR 75

proteins bind with high specificity to their intended endogenous RNA target in vivo. At the same 76

time, we demonstrate the utility of aPPRs for a particular application – the purification of specific 77

native ribonucleoprotein particles (RNPs) for identification of the associated proteins. The 78

population of proteins bound to an RNA influences its function and metabolism, but techniques 79

for characterizing RNP-specific proteomes are limited. Thus, our results expand the toolkit for 80

purifying selected RNPs and lay groundwork for the use of aPPRs in other applications. 81

82

RESULTS 83

We chose to target aPPRs to the chloroplast psbA mRNA for this proof-of-concept experiment 84

because the psbA mRNA exhibits dynamic changes in translation in response to light, and 85

identification of bound proteins may elucidate the underlying mechanisms (reviewed in Sun and 86

Zerges, 2015; Chotewutmontri and Barkan, 2018). We designed aPPR proteins with either 11 or 87

14 PPR motifs to bind the psbA 3ʹ-untranslated region (UTR) in Arabidopsis (Figure 1A, 88

Supplemental Figure 1). We refer to these proteins as SCD11 and SCD14, respectively. 89

Because PPR proteins bind single-stranded RNA, we targeted the proteins to a sequence that is 90

predicted to be unstructured. To avoid disrupting psbA expression, we chose a target sequence 91

that is poorly conserved and that begins sufficiently far from the stop codon that the terminating 92

ribosome and aPPR should not occupy the same nucleotides. We designed the proteins 93

according to the scheme described by Shen and coworkers (Shen et al., 2015), such that a tract 94

of consensus PPR motifs with the appropriate specificity-determining amino acids is embedded 95

within N- and C-terminal segments of the native chloroplast-localized protein PPR10 (Pfalz et 96

al., 2009). We previously reported a comprehensive analysis of the sequence specificity of 97

SCD11 and SCD14 in vitro (Miranda et al., 2018), which confirmed them to be highly selective 98

for their intended target sequence in vitro while also revealing nuances relevant to prediction of 99

off-target binding. For the in vivo assays described here, the proteins included, in addition, a C-100

terminal 3xFLAG tag and the N-terminal chloroplast targeting sequence from PPR10, which is 101

cleaved after chloroplast import (Figure 1A and Supplemental Figure 1). 102

Immunoblot analysis of leaf and chloroplast fractions from transgenic Arabidopsis plants 103

expressing SCD11 and SCD14 confirmed that they localize to chloroplasts (Figure 1B). Both 104

proteins were found predominantly in the soluble fraction, as expected given that they lack 105

transmembrane segments or thylakoid targeting signals. Laddering beneath the band 106

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corresponding to each full-length protein suggests that these artificial proteins are prone to 107

proteolysis in vivo. The transgenic plants were phenotypically normal (Figure 1C) and had 108

normal levels of PsbA protein (Figure 1B), indicating that the aPPRs did not disrupt psbA 109

expression or have off-target effects that compromised plant growth. 110

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SCD11 and SCD14 Bind with High Specificity to psbA RNA in vivo 112

To identify RNAs that are bound to SCD11 and SCD14 in vivo, we isolated chloroplasts from the 113

transgenic plants, used anti-FLAG antibody to immunoprecipitate each protein from stromal 114

extract (Figure 2A), and purified RNA from the coimmunoprecipitates. Slot-blot hybridizations 115

showed that psbA RNA was highly enriched in immunoprecipitates from the transgenic lines in 116

comparison to the wild-type progenitor (Col-0) (Figure 2B). Furthermore, RNA from the 3ʹ-UTR 117

was more highly enriched than that from the 5ʹ-UTR (Figure 2B), consistent with the binding of 118

the aPPRs to the 3ʹ-UTR, as intended. 119

To gain a comprehensive view of the RNAs bound to each protein, we sequenced the 120

coimmunoprecipitating RNA (RIP-seq) in two replicate experiments. Comparison of these RNAs 121

to those from immunoprecipitations with an antibody that does not recognize proteins in 122

Arabidopsis showed that the psbA RNA was strongly enriched in the SCD11 and SCD14 123

immunoprecipitates (Figure 2C, Supplemental Figure 2A, Supplemental Dataset 1). The 124

enrichment of RNAs mapping to each chloroplast protein-coding gene was highly reproducible 125

between the replicate experiments (Figure 2D and Supplemental Figure 2A); RNA from the 126

psbA gene was enriched more than 100-fold, whereas RNA from most genes showed no 127

enrichment. This establishes that artificial PPR proteins can bind specifically to an intended 128

RNA target in vivo. The 4.5S rRNA was also enriched in the SCD11 immunoprecipitates. 129

However, this may be artifactual because it was not enriched in SCD14 immunoprecipitates, 130

and 4.5S rRNA is an unlikely ligand for an aPPR protein due to the fact that it is highly 131

structured and largely embedded in the ribosome. 132

Reproducible low-level enrichment of RNA from several genes other than psbA 133

suggested a small degree of off-target binding, particularly by SCD14 (Figure 2D and 134

Supplemental Figure 2A). All genes whose RNAs showed greater than three-fold enrichment 135

in either the SCD14 or SCD11 coimmunoprecipitates are listed in Figure 3A. Most of these 136

contain local peaks of enrichment harboring sequences resembling the intended binding site in 137

the psbA 3ʹ-UTR (Figure 3B and Supplemental Figure 3). A sequence logo representing the 138

frequency of nucleotides at each position in SCD14’s off-target sequence set closely resembles 139

SCD14’s intended binding site (Figure 3C). The in vivo specificity landscapes of SCD11 and 140

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SCD14 implied by these data are discussed below, in the context of our prior in vitro bind-n-seq 141

analysis of the same proteins (Miranda et al., 2018). 142

143

Identification of Known and Novel psbA RNA-Binding Proteins in SCD14 and SCD11 144

Coimmunoprecipitates 145

Results above demonstrated that psbA RNA is, by far, the most highly enriched mRNA in 146

SCD14 and SCD11 coimmunoprecipitates. To identify native proteins that are bound to psbA 147

mRNA in vivo, we analyzed the SCD11 and SCD14 coimmunoprecipitates by mass 148

spectrometry. Approximately 400 different proteins were identified in at least one of the 149

immunoprecipitates (Supplemental Dataset 2). The enrichment of each protein was calculated 150

with respect to its representation in an anti-FLAG immunoprecipitate from the non-transgenic 151

host line (Col-0). Approximately 50 proteins were enriched at least two-fold in either the SCD11 152

or SCD14 immunoprecipitation (Supplemental Dataset 2). Proteins whose enrichment 153

averaged at least three-fold in the two experiments are listed in Figure 4A. This protein set 154

included several proteins that are known to associate with psbA mRNA: HCF173, which 155

activates psbA translation (Schult et al., 2007), cpSRP54, which binds cotranslationally to PsbA 156

(Nilsson and van Wijk, 2002), and various ribosomal proteins and general translation factors. 157

Immunoblot analysis of anti-FLAG coimmunoprecipitates confirmed that HCF173 and cpSRP54 158

coimmunoprecipitate with the aPPR proteins from extracts of the transgenic plants (Figure 4B). 159

The differing efficiency with which these proteins were coprecipitated from the two lines may be 160

due to the higher abundance of SCD14 in the stromal extracts (see Figure 1B), or to differing 161

degrees of RNA degradation in the two preparations, as RNA cleavage will separate the bound 162

aPPRs from proteins bound elsewhere on the RNA. 163

Notably absent from the set of proteins detected in the SCD11 and SCD14 164

immunoprecipitates (Supplemental Dataset 2) is the pentatricopeptide repeat protein LPE1, 165

which was reported to bind the psbA 5ʹ-UTR and recruit HCF173 for psbA translation (Jin et al., 166

2018). However, this role for LPE1 was recently called into question (Williams-Carrier et al., 167

2019). The failure to detect LPE1 in the aPPR coimmunoprecipitates despite the strong 168

enrichment of HCF173 supports the revised view that LPE1 neither binds psbA RNA nor 169

activates its translation. 170

Two predicted RNA-binding proteins of unknown function were strongly enriched in the 171

aPPR coimmunoprecipitates (Figure 4A): SRRP1 (AT3G23700) and CP33C (AT4G09040). 172

SRRP1 has two S1 RNA-binding domains and was proposed to harbor RNA chaperone activity 173

based on in vitro and E. coli complementation data (Gu et al., 2015). CP33C has two RRM 174

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RNA-binding domains (Ruwe et al., 2011), but functional studies have not been reported. To 175

determine whether these proteins associate with psbA RNA in vivo, we performed RIP-seq 176

using antibodies generated against the maize orthologs (sequences shown in Supplemental 177

Figure 4A). At the same time, we performed RIP-seq with antibody to the maize ortholog of 178

HCF173 (Zm-HCF173); HCF173 was previously shown to coimmunoprecipitate with psbA RNA, 179

but limited information was provided about interactions with other RNAs (Schult et al., 2007). 180

Because the Zm-CP33C antibodies did not detect the Arabidopsis ortholog, we used maize 181

stroma for this set of RIP-seq assays. The psbA RNA was highly enriched in each 182

coimmunoprecipitate and was the only RNA to be strongly enriched in each case (Figure 5, 183

replicates in Supplemental Figure 2B). Chloroplast rRNAs were reproducibly enriched in the 184

Zm-HCF173 and Zm-SRRP1 immunoprecipitates but not in the Zm-CP33C immunoprecipitates, 185

suggesting that HCF173 and SRRP1, but not CP33C, associate with ribosomes and/or with 186

psbA RNA that is undergoing translation. Several other mRNAs were reproducibly enriched in 187

each immunoprecipitate, but to a much smaller degree (Figure 5B, Supplemental Figure 2B). 188

These results validate the utility of the aPPR-affinity tag approach to identify proteins that 189

associate with a specific RNA-of-interest in vivo. 190

191

High-Resolution RIP-seq Pinpoints Binding Sites for HCF173 and CP33C on psbA mRNA 192

The precise location of an RNA-binding protein on RNA can be informative with regard to its 193

functions and mechanisms. With that in mind, we modified the RIP-seq protocol by adding a 194

Ribonuclease I digestion step prior to antibody addition, aiming to limit the span of RNA that 195

coimmunoprecipitates due to proximity to the binding site. The RNase treatment all but 196

eliminated psbA RNA from the SRRP1 coimmunoprecipitation (Figure 6A), suggesting that the 197

Zm-SRRP1 binding site is too short or of too low an affinity to be recovered with this method. 198

However, psbA sequences remained highly enriched in the Zm-HCF173 and Zm-CP33C 199

coimmunoprecipitates (Figure 6A). The apparent increase in rRNA abundance in the +RNase 200

immunoprecipitates likely reflects a decrease in the balance of true RNA ligand (psbA RNA) to 201

contaminants (rRNA), as we aimed to obtain a similar number sequence reads in all 202

experiments. A higher resolution view of these data showed that both antibodies 203

immunoprecipitated well-defined, specific fragments of psbA RNA (Figure 6B). The sequences 204

that coimmunoprecipitated with CP33C mapped toward the end of the open reading frame 205

(Figure 6B and Supplemental Figure 4B). The sequence that coimmunoprecipitated with 206

HCF173 mapped in the 5ʹ UTR (Figure 6B, red) and coincided with a conserved sequence 207

patch (Figure 6C). We refer to this as the HCF173 footprint, but recognize that HCF173’s 208

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interaction with this sequence could be mediated by another protein. Interestingly, this 209

sequence spans a junction between two predicted conserved RNA hairpins (Figure 6C). The 210

downstream hairpin intrudes on the footprint of the initiating ribosome (Chotewutmontri and 211

Barkan, 2016) and is therefore predicted to inhibit translation initiation (Scharff et al., 2011). 212

Association of HCF173 with the site defined here would likely prevent formation of this hairpin, 213

providing a plausible mechanism for HCF173’s translation activation function. 214

DISCUSSION 215

PPR proteins have attracted interest as potential scaffolds for the development of designer 216

RNA-binding proteins (Filipovska and Rackham, 2011; Yagi et al., 2014; Hall, 2016). However, 217

the feasibility of designing PPR proteins to bind specifically to a wide variety of RNA sequences, 218

and of predicting the landscape of RNA occupancy in vivo remain to be established. Results 219

presented here advance these efforts by demonstrating that artificial proteins built from 220

consensus PPR motifs bind with high specificity to the intended RNA target in vivo, that the 221

affinity of these interactions is sufficient for RNA-tagging applications, and that the landscape of 222

low-level off-target binding correlates well with data from in vitro experiments. 223

As is true of all RNA-binding proteins, the sequence specificity of PPR proteins is not 224

absolute: the population of bound RNAs will inevitably be influenced by protein concentration, 225

salt conditions, RNA structure, and competition from other RNAs. As expected, we observed a 226

small degree of off-target binding by our artificial proteins. Encouragingly, however, features of 227

these off-target interactions (Figure 3) closely resemble those from our prior in vitro bind-n-seq 228

analyses of the same proteins (Miranda et al., 2018). For example, SCD14 was more 229

permissive to mismatches in the binding site than was SCD11, nucleotide selectivity dropped off 230

toward the C-terminus, pyrimidine-binding motifs discriminated poorly between U and C, and 231

purine-binding motifs (especially those aligning with AGA at positions 5 through 7 of the binding 232

site) were the most highly selective. Additionally, sequences flanking the off-target sites are AU-233

rich (see logo in Figure 3C), implying that off-target binding in vivo is concentrated in regions of 234

low RNA structure. This is consistent with our finding that binding of a model PPR protein in 235

vitro is inhibited even by very weak RNA structures (McDermott et al., 2018). The fact that in 236

vitro bind-n-seq data were highly predictive of the in vivo interaction landscapes of our artificial 237

PPR proteins indicates that bind-n-seq analysis is a fruitful precursor to in vivo applications. 238

In addition, we show that the capability of aPPRs to bind predictably to RNA in vivo can 239

be used to characterize proteomes associated with specific RNAs. The panoply of proteins that 240

bind an RNA determine many aspects of its function and metabolism. Although excellent 241

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approaches are available for identifying RNAs bound to a protein of interest, the identification of 242

proteins bound to particular RNAs remains problematic. Several methods for the RNA-centric 243

purification of RNPs have been reported previously. Some of these rely on the insertion of an 244

RNA affinity tag into the target RNA (e.g. Butter et al., 2009; Panchapakesan et al., 2017; 245

Ramanathan et al., 2018). However, insertions can alter RNA functionalities, modification of 246

endogenous genes is technically challenging in some experimental systems (such as 247

organelles), and expression of ectopic modified genes can disrupt the balance of trans-factors 248

to their cis-targets. These limitations are addressed by assays that purify untagged RNPs by 249

coupling in vivo crosslinking with post-lysis antisense oligonucleotide purification (e.g. Chu et 250

al., 2015; McHugh et al., 2015; Rogell et al., 2017; Spiniello et al., 2018). However, UV 251

crosslinking is inefficient and is practical only with cultured cells or lysates. Formaldehyde 252

crosslinking provides an alternative, but is prone to capturing both transient and stable 253

interactions. Recently, a Type VI-related CRISPR-Cas system was engineered to bind and 254

modify the splicing of endogenous RNAs (Konermann et al., 2018). Whether this system can 255

achieve the degree of RNA occupancy needed for use in affinity purification approaches 256

remains to be determined. 257

Designer PPR affinity tags add to this toolkit by binding unmodified RNPs within intact 258

tissues, and allowing their purification under non-denaturing conditions. Given the successes in 259

using designer PUF proteins to modify the expression of specific mRNAs (Wang et al., 2009; 260

Campbell et al., 2014) and to track untagged RNAs in vivo (Yoshimura and Ozawa, 2016), they 261

may also be useful as affinity tags for RNP purification. However, the greater flexibility in repeat 262

tract length with the PPR scaffold (Hall, 2016) may facilitate customization of RNA binding 263

affinity, kinetics, and specificity for this application. 264

Our approach uncovered two RNA binding proteins of unknown function that associate 265

primarily with psbA RNA in vivo. It seems likely that these proteins influence psbA expression, 266

and elucidation of their functions will be an interesting area for future investigation. However, 267

there are several important caveats relevant to the general applicability of our approach. First, 268

the psbA mRNA is highly abundant, and analysis of proteomes associated with less abundant 269

mRNAs is likely to be more challenging. Second, PPR proteins in nature function almost 270

exclusively inside mitochondria and chloroplasts, and it is as yet unclear how they will perform in 271

the nuclear-cytosolic compartment. Thus, an important next step is to test this approach on a 272

cytosolic RNA target. Additionally, our finding that SCD14 and SCD11 are prone to proteolysis 273

in vivo warrants exploration of alternative consensus designs that are more robust to the in vivo 274

protease milieu. 275

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276

METHODS 277

Development of Transgenic Lines 278

Genes for SCD14 and SCD11 were codon-optimized for Arabidopsis and assembled by PCR 279

from several overlapping synthetic DNA fragments (IDT). The nucleotide and protein sequences 280

are provided in Supplemental Figure 1. They were designed with the PPR nucleotide 281

specificity codes described previously (Barkan et al., 2012; Miranda et al., 2018) and 282

summarized in Figure 1A. The PPR-encoding genes were inserted into a modified form of 283

pCambia1300 harboring the Superpromoter (Lee et al., 2007) to drive transgene expression 284

(inserted at the XbaI site of pCambia1300) and encoding a 3X-FLAG tag at the C-terminus of 285

the inserted ORF (a gift from Jie Shen and Zhizhong Gong, China Agricultural University). The 286

plasmids were used to transform Arabidopsis thaliana (ecotype Col-0) using the floral dip 287

method (Zhang et al., 2006). Lines were screened by immunoblotting for aPPR expression, and 288

those with the highest expression were used for further experiments. An additional transgenic 289

line was developed using the MCD14 protein design we reported previously (Miranda et al., 290

2018); however, MCD14 transgenic lines failed to express the protein. 291

Plant Growth 292

Arabidopsis seeds were sterilized by incubation for 10 min in a solution containing 1% bleach 293

and 0.1% SDS, followed by a 70% ethanol wash. The seeds were then washed three times 294

with sterile water. Seeds were plated and grown in tissue culture dishes containing MS agar 295

medium: 4.33 g/L Murashige and Skoog basal salt medium (Sigma), 2% sucrose, 0.3% 296

Phytagel, pH 5.7. Transgenic plants were selected by the addition of 50 µg/mL hygromycin to 297

the growth medium. Plants used for chloroplast isolation and immunoprecipitation assays were 298

grown in a growth chamber in diurnal cycles (10 h light at 120 µmol photons m-2 s-1, 14 h dark, 299

22˚C) for 14 days. Arabidopsis was grown using cool white, high output fluorescent lamps 300

(F48T12/CW/HO, Sylvania). 301

Maize was grown and used for the preparation of stromal extracts using methods 302

described previously (Schmitz-Linneweber at al, 2005). Maize insertion alleles for Zm-cp33C 303

(GRMZM2G023591) and Zm-srrp1 (GRMZM2G016084) were obtained from the UniformMu 304

collection: the GRMZM2G023591 mutant corresponds to line (mu1032521, UFMu-02565); 305

the GRMZM2G016084 mutant corresponds to line (mu1076060, UFMu-09028). Positions of the 306

insertions are diagrammed in Supplemental Figure 5. 307

308

Chloroplast Isolation and Fractionation 309

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Maize chloroplast stroma for use in RIP-seq assays was prepared as described previously 310

(Ostheimer et al., 2003). Arabidopsis chloroplast stroma was prepared from chloroplasts 311

isolated from the aerial portion of 2-week old seedlings (40 g tissue) as described (Kunst, 1998), 312

with the following modifications: seedlings were not placed in ice water before homogenization, 313

sorbitol concentration in the homogenization buffer was reduced to 0.33 M, and plants were 314

homogenized in a blender using three 5-second bursts. Purified chloroplasts were resuspended 315

and lysed in Hypotonic Lysis Buffer (30 mM HEPES-KOH pH 8.0, 10 mM MgOAc-4H2O, 60 mM 316

KOAc, 2 mM DTT, 2 µg/mL aprotinin, 2 µg/mL leupeptin, 1 µg/mL pepstatin A, 0.8 mM PMSF), 317

using a minimal buffer volume. Lysed chloroplasts were centrifuged for 40 min at 18,000 x g at 318

4˚C in a tabletop microcentrifuge to pellet membranes and particulates. The supernatant was 319

removed, and the pellet was resuspended in Hypotonic Lysis Buffer and centrifuged again 320

under the same conditions. Supernatants were combined, aliquoted, and frozen at -80 ˚C. The 321

thylakoid membranes (pellet fraction) were aliquoted and frozen at -80 ˚C. 322

Antibodies, SDS-PAGE and Immunoblot Analysis 323

SDS-PAGE and immunoblot analyses were performed as described previously (Barkan, 1998). 324

A mouse monoclonal anti-FLAG M2 antibody was purchased from Sigma (F1804-1 MG, LOT 325

SLBW3851. The SRP54 antibody was a generous gift of Masato Nakai (Osaka University). Cox 326

II and actin antisera were purchased from Agrisera (AS04 053A and AS13 2640, respectively). 327

Polyclonal antibodies were raised in rabbits to recombinant fragments of the maize orthologs of 328

HCF173, AT4G09040/CP33C and AT3G23700/SRRP1; these correspond to maize genes 329

GRMZM2G397247, GRMZM2G023591, and GRMZM2G016084, respectively (see http://cas-330

pogs.uoregon.edu/#/ for evidence of orthology). The amino acids used for the HCF173, S1 and 331

RRM protein antigens and evidence for the specificity of the resulting antisera are shown in 332

Supplemental Figure 4 and Supplemental Figure 5. Maize CRP1 antibody (Fisk et al., 1999) 333

was used as the negative control for immunoprecipitations from Arabidopsis extract because it 334

does not interact with proteins in Arabidopsis. Antibodies used for immunoprecipitations were 335

affinity purified against their antigen prior to use. 336

Coimmunoprecipitation Experiments 337

Immunoprecipitation for analysis of proteins by mass spectrometry was performed as described 338

previously (Watkins et al., 2007) with minor modifications. In brief, experiments used 339

Arabidopsis stromal extract, anti-FLAG antibodies were crosslinked to magnetic Protein A/G 340

beads (Pierce), the beads were pre-washed in CoIP Buffer (20 mM Tris-HCl, pH 7.5, 150 mM 341

NaCl, 1 mM EDTA, 0.5% NP-40, 5 µg/mL aprotinin), and the antibody cross-linked beads were 342

titrated to determine the amount required to deplete the aPPR from the stromal extract. Stromal 343

http://cas-pogs.uoregon.edu/#/

http://cas-pogs.uoregon.edu/#/

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extract (400 µL at 6 mg protein/ml) was supplemented with RNAsin (Promega) to a 344

concentration of 1 unit/µL and precleared by centrifugation for 10 min at 18,000 x g at 4˚C. The 345

supernatant was removed to a new tube, antibody-bound beads were added and the mixture 346

was incubated at 4˚C for one hour while rotating. Beads were captured with a magnet 347

(Invitrogen) and the supernatant was removed. The beads (pellets) were washed three times 348

with CoIP Buffer and then twice with 50 mM ammonium bicarbonate (pH 7.5). Proteins were 349

digested on the beads with trypsin (Promega mass spectrometry grade at 25 ng/µL in 50 mM 350

ammonium bicarbonate pH 7.5) overnight at 25˚C while shaking. Beads were captured and the 351

supernatant was transferred to a new tube. This step was repeated five times to ensure the 352

removal of all beads. LC-MS/MS was performed by the UC Davis Proteomics Core Facility, 353

where the data were analyzed using Scaffold2 (Proteome Software Inc). Protein enrichment 354

was calculated by dividing the average Normalized Spectral Abundance Factor (NSAF) values 355

(Zhang et al., 2010) from the two aPPR lines by NSAF values from the control 356

immunoprecipitation using extract of Col-0 plants. To avoid division by zero, a correction term of 357

0.001 was added to each NSAF value in the control; therefore, the actual enrichment of proteins 358

that were not detected in the control is under-estimated. The data are shown in Supplemental 359

Dataset 2. 360

Immunoprecipitations for RIP-seq analysis were performed similarly, except that 361

antibodies were not crosslinked to the beads, the Arabidopsis experiments used 200 µl of 362

extract, and the maize experiments used 70 µl of stromal extract at ~10 mg protein/ml and did 363

not include RNAsin. Antibody to maize CRP1 was used as a negative control for the aPPR RIP-364

seq assays; this antibody does not recognize proteins in Arabidopsis chloroplasts. The aPPR 365

RIP-seq experiments were performed two times, using the same stromal extracts. Antibody to 366

AtpB (subunit of the chloroplast ATP synthase) was used as the negative control for the RIP-367

seq assays using maize stroma. The HCF173, CP33C, and SRRP1 RIP-seq experiments were 368

each performed twice, using antibodies from different rabbits. For high-resolution RIP-seq, 369

stromal extract was pre-treated with 1U/µl RNAse I (Ambion) at 25˚C for 10 min. The sample 370

was placed on ice and the remainder of the procedure was as described above for standard 371

RIP-seq. 372

373 Analysis of Coimmunoprecipitated RNA by RIP-seq and Slot-blot Hydridization 374

An equal volume of TESS Buffer (10 mM Tris pH 7.5, 1 mM EDTA, 150 mM NaCl, 0.2% SDS) 375

supplemented with Proteinase K (0.2 g/µL) was added to the supernatant and pellet fractions 376

and incubated for 30 min at 37˚C. RNA was then purified by phenol-chloroform extraction and 377

12

ethanol precipitation, resuspended in H2O, and quantified by Qubit. The RNA was used directly 378

for slot-blot hybridizations as previously described (Schmitz-Linneweber et al., 2005), or 379

phosphorylated (T4 polynucleotide kinase, NEB) and processed for sequencing using the BIOO 380

Scientific NEXTflex Small RNA-Seq Kit v3 (Cat# NOVA-5132-06). For Arabidopsis, 50 ng of 381

pellet RNA was used as the input for sequencing libraries. The maize experiments used 20 ng 382

of pellet RNA for library preparation, and included an RNA fragmentation step: RNA was 383

fragmented by incubation at 95˚C for 4 min in 40 mM Tris-Acetate pH 8, 100 mM KOAc, 30 mM 384

Mg(OAc)2. The reaction was stopped by the addition of EDTA to a final concentration of 50 mM, 385

the RNA was ethanol precipitated in the presence of 1.5 µg GlycoBlue (Thermo Fisher), and 386

phosphorylated (T4 polynucleotide kinase, NEB) prior to library preparation. RNase I-RIP-seq 387

experiments did not include an RNA fragmentation step. Libraries were gel purified to enrich for 388

inserts between 15 and 100 nucleotides. Libraries were sequenced at the University of Oregon 389

Genomics and Cell Characterization Core Facility, with read lengths of 75 or 100 nucleotides. 390

Sequencing data were processed as in (Chotewutmontri and Barkan, 2018) except that all read 391

lengths were included and reads were aligned only to the chloroplast genome. Read counts for 392

RIP-seq experiments are summarized in Supplemental Dataset 1. Enrichment values were 393

calculated as the normalized abundance of reads mapping to each gene (including UTRs) in an 394

experimental sample relative to the control. 395

396

Accession Numbers 397

HCF173, AT1G16720; Zm-HCF173, Zm00001d014716_T002 (B73 v4) or 398

GRMZM2G397247_T03 (B73 v3); SRRP1, AT3G23700; Zm-SRRP1, GRMZM2G016084_T02 399

(B73 v3) or Zm00001d034828 (B73 v4); CP33C, AT4G09040; Zm-CP33C, 400

GRMZM2G023591_T01 (B73 v4) or Zm00001d031258_T005 (Bs3 v4) 401

402

Supplemental Data 403

Supplemental Figure 1. Sequences of SCD14 and SCD11. 404

Supplemental Figure 2. Replicate RIP-seq data for SCD14, SCD11, HCF173, SRRP1, and 405

CP33C 406

Supplemental Figure 3. High resolution views of RNA enrichment along genes listed in Figure 407

3A. 408

Supplemental Figure 4. Maize CP33C and SRRP1 antigens and CP33C RNA footprint. 409

Supplemental Figure 5. Additional information to support HCF173, CP33C, and SRRP1 RIP-410

seq. 411

13

Supplemental Dataset 1. Read counts and RPKM values for RIP-seq experiments. 412

Supplemental Dataset 2. Proteins found in SCD11 and SCD14 coimmunoprecipitates as 413

detected by LC-MS/MS. 414

415

Acknowledgments 416

We are grateful to Jie Shen (Chinese Academy of Sciences) and Zhizhong Gong (China 417

Agricultural University) for advice and for their gift of the pCAMBIA1300 vector modified to 418

encode a FLAG tag. We are also grateful to Masato Nakai (Osaka University) for the gift of 419

cpSRP54 antibody, Carolyn Brewster, Margarita Rojas, and Susan Belcher for technical 420

assistance, the UniformMu project (University of Florida) for maize insertion lines, and the UC-421

Davis Proteomics Core for LC-MS/MS proteomic analyses. This work was supported by 422

National Science Foundation grant MCB-1616016 (to A.B.) and National Institutes of Health 423

Training Grant T32-GM007759 (to J.J.M.). 424

425

AUTHOR CONTRIBUTIONS 426

A.B. and J.M. conceived the project and designed the experiments. J.M., R.W.-C., and K.W. 427

performed the experiments. All authors analyzed the data. A.B. and J.M. wrote the article. 428

429

References 430

431

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587

588

17

Figure Legends 589

590

Figure 1. Overview of artificial PPR proteins designed to bind the psbA 3ʹ UTR. 591

(A) Protein design. SCD11 and SCD14 were designed to bind the indicated 14 or 11 592

(underlined) nucleotide sequence in the 3ʹ-UTR of the psbA mRNA in Arabidopsis. The targeted 593

sequence begins 13 nucleotides downstream of the stop codon and ends five (SCD14) or eight 594

(SCD11) nucleotides upstream of the 3ʹ-terminal stem-loop in the psbA mRNA. SCD14 and 595

SCD11 contain 13 and 10 consensus PPR motifs, respectively, flanked by sequences from 596

PPR10 (green). The motifs that are found in SCD14 but not in SCD11 are marked in gray. The 597

specificity-determining amino acids (Barkan et al., 2012) (positions 5 and 35 according to the 598

nomenclature in (Yin et al., 2013) are indicated, and each repeat is aligned with its nucleotide 599

ligand. The PPR10-derived sequence at the N-terminus includes a chloroplast targeting 600

sequence and PPR10’s first PPR motif, which has a non-canonical specificity code (dotted line). 601

The targeting sequence is cleaved after import into the chloroplast (scissors). Both proteins 602

contain a C-terminal 3x FLAG tag. 603

(B) Immunoblots demonstrating chloroplast-localization of SCD11 and SCD14. Chloroplasts 604

(Cp) were isolated from total leaf (T) of wild-type (Col-0) and transgenic Arabidopsis plants, and 605

fractionated to generate thylakoid membrane (Th) and soluble (S) fractions. Aliquots 606

representing an equivalent amount of starting material were probed to detect markers for 607

cytosol (Actin), mitochondria (CoxII), and thylakoid membranes (PsbA). The aPPR proteins 608

were detected with anti-FLAG antibody. The Ponceau S-stained filter is shown below to 609

demonstrate the partitioning of the chloroplast stromal protein RbcL among the fractions. 610

(C) Visible phenotype of transgenic Arabidopsis plants expressing SCD14 and SCD11. Col-0 is 611

the wild-type progenitor of the transgenic lines. 612

613

614

18

Figure 2. RIP-seq analysis of RNAs that coimmunoprecipitate with SCD14 and SCD11. 615

(A) Immunoprecipitation of SCD14 and SCD11. Stromal extracts from transgenic Arabidopsis 616

expressing the indicated protein or from the Col-0 progenitor were used for immunoprecipitation with 617

anti-FLAG antibody. The pellet (P) and supernatant (S) fractions were analyzed by immunoblot 618

analysis using anti-FLAG antibody. An excerpt of the Ponceau S-stained filter is shown to illustrate 619

the abundance of the large subunit of Rubisco (RbcL), which serves as a loading control. An equal 620

proportion of each pellet fraction was analyzed; 1/4th

that proportion of each supernatant was 621

analyzed to avoid overloading the lane. 622

(B) Slot blot hybridization analysis of RNAs that coimmunoprecipitate with SCD14 and SCD11. RNA 623

was extracted from the immunoprecipitations analyzed in (A) and applied to nylon membrane via a 624

slot-blot manifold. The same proportion of all samples was analyzed to illustrate the partitioning of 625

psbA RNA between the pellet and supernatant fractions. Replicate blots were hybridized with 626

oligonucleotide probes specific for the psbA 5ʹ-UTR or 3ʹ-UTR. 627

(C) Plastome-wide view of RNAs that coimmunoprecipitate with SCD11 and SCD14. Results are 628

plotted as the sequence coverage in consecutive 10-nt windows along the chloroplast genome 629

(Accession NC_000932.1) per million reads mapped to the chloroplast genome. The negative 630

control used an antibody that does not detect proteins in Arabidopsis together with extract from the 631

SCD14 line. Data for replicate experiments are shown in Supplemental Figure 2A. Read counts for 632

all RIP-seq experiments are provided in Supplemental Dataset 1. 633

(D) Enrichment of RNA from each protein-coding chloroplast gene in SCD11 and SCD14 634

coimmunoprecipitates. The ratio of normalized reads/gene in the experimental versus control 635

immunoprecipitations is shown for Replicate 1. Data for replicate experiments are shown in 636

Supplemental Figure 2A. 637

638

639

Figure 3. Analysis of off-target binding by SCD14 and SCD11. 640

(A) Genes whose transcripts were enriched more than three-fold in both replicate SCD14 and 641

SCD11 coimmunoprecipitates. The position of peak enrichment within each gene (see panel B 642

and Supplemental Figure 3) and the magnitude of enrichment at that peak (average of 643

replicates) are indicated, together with sequence motifs within the peak that resemble the 644

intended binding site for SCD14 and SCD11. Matches to the intended binding site in the psbA 645

3ʹ-UTR are shaded in black. Transition mismatches are shaded in gray. The nucleotide at the 646

peak of enrichment (see profiles in Figure 3B and Supplemental Figure 3) is marked in red. 647

(B) Local sequence enrichment profile for the off-target sites in rpl32 and ccsA. The regions 648

shown span the ORF and UTRs of the indicated gene. The displayed sequences overlap the 649

point of maximum enrichment (nucleotide at peak marked in red) and resemble the intended 650

binding site of SCD14 (see panel A). Analogous plots for other genes listed in panel (A) are 651

shown in Supplemental Figure 3. 652

(C) Sequence logo derived from the off-target sites of SCD14. Sequences listed in panel (A) 653

were weighted according to the degree to which RNA from the corresponding region was 654

enriched in the SCD14 immunoprecipitation, and analyzed with WebLogo (Crooks et al., 2004). 655

656

657

19

Figure 4. Highly-enriched proteins in SCD11 and SCD14 coimmunoprecipitates. 658

(A) Proteins whose average enrichment from lines expressing SCD11 or SCD14 in comparison659

to the Col-0 progenitor was three or greater. Stars mark two RNA-binding proteins of unknown660

function. HCF173 (hashtag) is known to associate with psbA mRNA and to activate its661

translation (Schult et al., 2007; Williams-Carrier et al., 2019). The full dataset is provided in662

Supplemental Dataset 2, which includes a more complete explanation of the data analysis.663

(B) Immunoblot validation of two proteins identified by MS analysis. Chloroplast stroma from664

plants expressing SCD11 or SCD14, or from the Col-0 progenitor was used for665

immunoprecipitation with anti-FLAG antibody. Replicate immunoblots were probed with anti-666

FLAG antibody to detect SCD11 and SCD14 (top panel), HCF173, or cpSRP54. The HCF173667

blot was initially probed to detect RbcL, an abundant protein that typically contaminates668

immunoprecipitates; this serves as an internal standard.669

670

Figure 5. RIP-Seq analysis of Zm-HCF173, Zm-CP33C and Zm-SRRP1. 671

Maize chloroplast stroma was used for immunoprecipitations with antibodies to the maize 672

orthologs of HCF173, CP33C, and SRRP1, and the coimmunoprecipitated RNA was analyzed 673

by deep sequencing. 674

(A) Average sequence coverage in consecutive 10-nt windows along the chloroplast genome,675

per million reads mapped to the chloroplast genome (NCBI NC_001666).676

(B) Ratio of normalized reads/gene in the experimental immunoprecipitations versus a control677

using antibody to AtpB. Analogous plots for replicate experiments are shown in Supplemental678

Figure 2B. Read counts are provided in Supplemental Dataset 1, which also includes data for679

tRNAs. Immunoblots demonstrating immunoprecipitation of each protein are shown in680

Supplemental Figure 5.681

682

683

Figure 6. High resolution RIP-seq analysis of HCF173 and CP33C pinpointing interaction sites 684

in the psbA mRNA. Experiments were performed as in Figure 5 except that the stromal extract 685

was briefly treated with RNase I prior to antibody addition. 686

(A) Average sequence coverage in consecutive 10-nt windows along the chloroplast genome,687

per million reads mapped to the chloroplast genome (NCBI NC_001666).688

(B) Sequence coverage along the psbA mRNA, with (red, + RNase) or without (black, -RNase)689

a pre-treatment of stroma with RNase I. The number of reads per million mapped to the690

chloroplast genome (Y axes) is shown according to position along the chloroplast genome (X691

axes). The sequences of the RNase-resistant peaks are shown in panel (C) (HCF173) and692

Supplemental Figure 4B (CP33C).693

(C) Site of HCF173 interaction in the psbA 5ʹ UTR. The RNase-resistant sequence that694

coimmunoprecipitates with HCF173 (HCF173 footprint) is marked on a multiple sequence695

alignment of the psbA 5ʹ UTR from Arabidopsis, maize, tobacco, and rice. A conserved696

secondary structure was predicted by Dynalign (Fu et al., 2014) using the maize and697

Arabidopsis sequences as input; the prediction for maize is shown below.698

699

700

1

Figure 1. Overview of artificial PPR proteins designed to bind the psbA 3’ UTR. (A) Protein design. SCD11 and SCD14 were designed to bind the indicated 14 or 11(underlined) nucleotide sequence in the 3’-UTR of the psbA mRNA in Arabidopsis. The targetedsequence begins 13 nucleotides downstream of the stop codon, and ends five (SCD14) or eight(SCD11) nucleotides upstream of the 3’-terminal stem-loop in the psbA mRNA. SCD14 andSCD11 contain 13 and 10 consensus PPR motifs, respectively, flanked by sequences fromPPR10 (green). The motifs that are found in SCD14 but not in SCD11 are marked in gray. Thespecificity-determining amino acids (Barkan et al., 2012) (positions 5 and 35 according to thenomenclature in (Yin et al., 2013) are indicated, and each repeat is aligned with its nucleotideligand. The PPR10-derived sequence at the N-terminus includes a chloroplast targetingsequence and PPR10’s first PPR motif, which has a non-canonical specificity code (dotted line).The targeting sequence is cleaved after import into the chloroplast (scissors). Both proteinscontain a C-terminal 3x FLAG tag.(B) Immunoblots demonstrating chloroplast-localization of SCD11 and SCD14. Chloroplasts(Cp) were isolated from total leaf (T) of wild-type (Col-0) and transgenic Arabidopsis plants, andfractionated to generate thylakoid membrane (Th) and soluble (S) fractions. Aliquotsrepresenting an equivalent amount of starting material were probed to detect markers forcytosol (Actin), mitochondria (CoxII), and thylakoid membranes (PsbA). The aPPR proteinswere detected with anti-FLAG antibody. The Ponceau S-stained filter is shown below todemonstrate the partitioning of the chloroplast stromal protein RbcL among the fractions.(C) Visible phenotype of transgenic Arabidopsis plants expressing SCD14 and SCD11. Col-0 isthe wild-type progenitor of the transgenic lines.

2

Figure 2. RIP-seq analysis of RNAs that coimmunoprecipitate with SCD14 and SCD11. (A) Immunoprecipitation of SCD14 and SCD11. Stromal extracts from transgenic Arabidopsisexpressing the indicated protein or from the Col-0 progenitor were used for immunoprecipitation withanti-FLAG antibody. The pellet (P) and supernatant (S) fractions were analyzed by immunoblotanalysis using anti-FLAG antibody. An excerpt of the Ponceau S-stained filter is shown to illustratethe abundance of the large subunit of Rubisco (RbcL), which serves as a loading control. An equalproportion of each pellet fraction was analyzed; 1/4th that proportion of each supernatant wasanalyzed to avoid overloading the lane.(B) Slot blot hybridization analysis of RNAs that coimmunoprecipitate with SCD14 and SCD11. RNAwas extracted from the immunoprecipitations analyzed in (A) and applied to nylon membrane via aslot-blot manifold. The same proportion of all samples was analyzed to illustrate the partitioning ofpsbA RNA between the pellet and supernatant fractions. Replicate blots were hybridized witholigonucleotide probes specific for the psbA 5’-UTR or 3’-UTR.(C) Plastome-wide view of RNAs that coimmunoprecipitate with SCD11 and SCD14. Results areplotted as the sequence coverage in consecutive 10-nt windows along the chloroplast genome(Accession NC_000932.1) per million reads mapped to the chloroplast genome. The negativecontrol used an antibody that does not detect proteins in Arabidopsis together with extract from theSCD14 line. Data for replicate experiments are shown in Supplemental Figure 2A. Read counts forall RIP-seq experiments are provided in Supplemental Table 1.(D) Enrichment of RNA from each protein-coding chloroplast gene in SCD11 and SCD14coimmunoprecipitates. The ratio of normalized reads/gene in the experimental versus controlimmunoprecipitations is shown for Replicate 1. Data for replicate experiments is shown inSupplemental Figure 2A.

3

Figure 3. Analysis of off-target binding by SCD14 and SCD11. (A) Genes whose transcripts were enriched more than three-fold in both replicate SCD14 andSCD11 coimmunoprecipitates. The position of peak enrichment within each gene (see panel Band Supplemental Figure 3) and the magnitude of enrichment at that peak (average ofreplicates) are indicated, together with sequence motifs within the peak that resemble theintended binding site for SCD14 and SCD11. Matches to the intended binding site in the psbA 3’UTR are shaded in black. Transition mismatches are shaded in gray. The nucleotide at the peakof enrichment (see profiles in Figure 3B and Supplemental Figure 3) is marked in red.(B) Local sequence enrichment profile for the off-target sites in rpl32 and ccsA. The regionsshown span the ORF and UTRs of the indicated gene. The displayed sequences overlap thepoint of maximum enrichment (nucleotide at peak marked in red) and resemble the intendedbinding site of SCD14 (see panel A). Analogous plots for other genes listed in panel (A) areshown in Supplemental Figure 3.(C) Sequence logo derived from the off-target sites of SCD14. Sequences listed in panel (A)were weighted according to the degree to which RNA from the corresponding region wasenriched in the SCD14 immunoprecipitation, and analyzed with WebLogo (Crooks et al., 2004).

4

Figure 4. Highly-enriched proteins in SCD11 and SCD14 coimmunoprecipitates. (A) Proteins whose average enrichment from lines expressing SCD11 or SCD14 in comparisonto the Col-0 progenitor was three or greater. Stars mark two RNA-binding proteins of unknownfunction. HCF173 (hashtag) is known to associate with psbA mRNA and to activate itstranslation (Schult et al., 2007; Williams-Carrier et al., 2019). The full dataset is provided inSupplemental Table 2, which includes a more complete explanation of the data analysis.(B) Immunoblot validation of two proteins identified by MS analysis. Chloroplast stroma fromplants expressing SCD11 or SCD14, or from the Col-0 progenitor was used forimmunoprecipitation with anti-FLAG antibody. Replicate immunoblots were probed with anti-FLAG antibody to detect SCD11 and SCD14 (top panel), HCF173, or cpSRP54. The HCF173blot was initially probed to detect RbcL, an abundant protein that typically contaminatesimmunoprecipitates; this serves as an internal standard.

5

Figure 5. RIP-Seq analysis of Zm-HCF173, Zm-CP33C and Zm-SRRP1. Maize chloroplast stroma was used for immunoprecipitations with antibodies to the maize orthologs of HCF173, CP33C, and SRRP1, and the coimmunoprecipitated RNA was analyzed by deep sequencing. (A) Average sequence coverage in consecutive 10-nt windows along the chloroplast genome,per million reads mapped to the chloroplast genome (NCBI NC_001666).(B) Ratio of normalized reads/gene in the experimental immunoprecipitations versus a controlusing antibody to AtpB. Analogous plots for replicate experiments are shown in SupplementalFigure 2B. Read counts are provided in Supplemental Table 1, which also includes data fortRNAs. Immunoblots demonstrating immunoprecipitation of each protein are shown inSupplemental Figure 5.

6

Figure 6. High resolution RIP-seq analysis of HCF173 and CP33C pinpointing interaction sites in the psbA mRNA. Experiments were performed as in Figure 5 except that the stromal extract was briefly treated with RNase I prior to antibody addition. (A) Average sequence coverage in consecutive 10-nt windows along the chloroplast genome,per million reads mapped to the chloroplast genome (NCBI NC_001666).(B) Sequence coverage along the psbA mRNA, with (red, + RNase) or without (black, -RNase)a pre-treatment of stroma with RNase I. Y-axes show the number of reads per million readsmapped to the chloroplast genome coordinate shown on the X axes. The sequences of theRNase-resistant peaks are shown in panel (C) (HCF173) and Supplemental Figure 4B(CP33C).(C) Site of HCF173 interaction in the psbA 5’ UTR. The RNase-resistant sequence thatcoimmunoprecipitates with HCF173 (HCF173 footprint) is marked on a multiple sequencealignment of the psbA 5’ UTR rom Arabidopsis, maize, tobacco, and rice. A conservedsecondary structure was predicted by Dynalign (Fu et al., 2014) using the maize andArabidopsis sequences as input; the prediction for maize is shown below.

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DOI 10.1105/tpc.19.00177; originally published online May 23, 2019;Plant Cell

James J McDermott, Kenneth P Watkins, Rosalind Williams-Carrier and Alice BarkanArabidopsis

Ribonucleoprotein capture by in vivo expression of a designer pentatricopeptide repeat protein in

This information is current as of March 20, 2020

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