The Chloroplast Ribonucleoprotein CP33B Quantitatively Binds the psbA mRNA
Abstract
:1. Introduction
2. Results
2.1. CP33B Localizes to the Chloroplast
2.2. CP33B Has a Preference for the psbA mRNA Over Other Chloroplast Transcripts
2.3. CP33B Quantitatively Co-immunoprecipitates psbA mRNAs
2.4. CP33B Prefers an RNA Sequence Motif in Vitro
2.5. Membrane-bound CP33B is Also Associated with Chloroplast mRNAs
3. Discussion
3.1. CP33B is a Global RBP With a Clear Preference for and Ability to Sequester the psbA mRNA Pool
3.2. cpRNPs Interact With Their Target Transcripts via Multiple Binding Sites
4. Materials and Methods
4.1. Plant Material
4.2. Localization by Fluorescence Microscopy
Oligonucleotide | Sequence |
33B.fw.xhoI.TP | ATACTCGAGATGGCGGTTTTGGAAGC |
33B.rev.ncoI.cDNA | ATACCATGGCCACAATGTTTTCTTCG |
4.3. Dot-Blot Analysis
Oligo Name | Sequence | Target Gene |
CK_atpH5‘_for | GATTTAGATAGGGATTCGATTAG | atpH |
CK_atpH5‘_T7_rev | GTAATACGACTCACTATAGGGTCCAGGTCCAATAGAAGCAAG | atpH |
Ck_psbD_for | CTATTTCGCTTTAGGGGGTTGG | psbD |
Ck_psbD_T7_rev | TAATACGACTCACTATAGGGCAGCAATGGGACCAGAGAATGC | psbD |
CK_rbcL_for | GCAGCATTCCGAGTAACTCC | rbcL |
CK_rbcL_T7 | GTAATACGACTCACTATAGGGCCACGTAGACATTCATAAACTGC | rbcL |
CK-petB-for | CGTCCAACCGTTACTGAAGC | petB |
CK-petB-T7-rev | GTAATACGACTCACTATAGGGAATAGCGTCAGGTACACC | petB |
CK-psbF-for | GTCTGGAAGCACAGGAGAACG | psbF |
CK-psbF-T7-rev | GTAATACGACTCACTATAGGGCAAAACGGCCTGTTATTAATGG | psbF |
ndhBex1.rp | CCGATGGAGAGAAGAACCTATG | ndhB |
ndhBex1.T7 | GTAATCGACTCACTATAGGGTAGCCAAGAGAAACCATGAACC | ndhB |
ndhK.rp | CTATGGCCGCTTCTTTATGG | ndhK |
ndhK.T7 | GTAATCGACTCACTATAGGGATTTCCAATATGCGGACTGC | ndhK |
psbA.rp | CATTCATTGCTGCTCCTCCAG | psbA |
psbA.t7neu | GTAATCGACTCACTATAGGGATTCCTAGAGGCATACCATCAG | psbA |
psbC.rp | AGTGGCCCATTTTGTACCTG | psbC |
psbC.T7 | GTAATCGACTCACTATAGGGCCCCCAAAGGGAGATTTTAG | psbC |
rrn23.rp | CCTAGATGGCGAGAGTCCAG | rrn23 |
rrn23.T7 | GTAATCGACTCACTATAGGGAAGACTCGCTTTCGCTACG | rrn23 |
4.4. Immunoblots and Antibody Production
4.5. RIP-chip Analysis
4.6. RBNS Analysis
4.6.1. Cloning of GST:SBP:CP33B
Oligonucleotide | Sequence |
CP33BXhoIrev | AATTCTCGAGTCACTCCACAATGTTTTCTTCGG |
MfeICP33Bfor | AATCAATTGGTCTCCGTACTCTGTTCCGTC |
4.6.2. RNA Oligo Production
Oligonucleotide | Sequence |
RBNS T7 Template | CCTTGACACCCGAGAATTCCA -(N)40- GATCGTCGGACTGTAGAACTCCCTATAGTGAGTCGTATTA |
RNA PCR (RP1) | AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACGATC |
RT primer | GCCTTGGCACCCGAGAATTCCA |
T7 Promoter Oligo | TAATACGACTCACTATAGGG |
4.6.3. RBNS Assay
Sample | NEXTflex Barcode Primer Sequence |
Input | 5 ′-CAAGCAGAAGACGGCATACGAGAT- CGTGATGTGACTGGAGTTCAGACGT- GTGCTCTTCCGATC-s-T-3 ′ |
0 protein sample | 5 ′- CAAGCAGAAGACGGCATACGAGA- TGTAGCCGTGACTGGAGTTCAGACGT- GTGCTCTTCCGATC-s-T- |
100 nM CP33B | 5 ′-CAAGCAGAAGACGGCATACGAGA- TCTGATCGTGACTGGAGTTCAGACGT- GTGCTCTTCCGATC-s-T-3 ′ |
1000 nM CP33B | 5 ′-CAAGCAGAAGACGGCATACGAGAT- AAGCTAGTGACTGGAGTTCAGACGT- |
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Teubner, M.; Lenzen, B.; Espenberger, L.B.; Fuss, J.; Nickelsen, J.; Krause, K.; Ruwe, H.; Schmitz-Linneweber, C. The Chloroplast Ribonucleoprotein CP33B Quantitatively Binds the psbA mRNA. Plants 2020, 9, 367. https://doi.org/10.3390/plants9030367
Teubner M, Lenzen B, Espenberger LB, Fuss J, Nickelsen J, Krause K, Ruwe H, Schmitz-Linneweber C. The Chloroplast Ribonucleoprotein CP33B Quantitatively Binds the psbA mRNA. Plants. 2020; 9(3):367. https://doi.org/10.3390/plants9030367
Chicago/Turabian StyleTeubner, Marlene, Benjamin Lenzen, Lucas Bernal Espenberger, Janina Fuss, Jörg Nickelsen, Kirsten Krause, Hannes Ruwe, and Christian Schmitz-Linneweber. 2020. "The Chloroplast Ribonucleoprotein CP33B Quantitatively Binds the psbA mRNA" Plants 9, no. 3: 367. https://doi.org/10.3390/plants9030367