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1. Two ATAF transcription factors ANAC102 and ATAF1 contribute to the suppression of cytochrome P450 ‐mediated brassinosteroid catabolism in Arabidopsis

   https://doi.org/10.1111/ppl.13339  

   Peng, Hao ; Neff, Michael M. ( February 2021 , Physiologia Plantarum)

   PHYB ACTIVATION TAGGED SUPPRESSOR 1 (BAS1) and SUPPRESSOR OF PHYB‐4 7 (SOB7) are two cytochrome P450 enzymes that inactivate brassinosteroids (BRs) inArabidopsis. The NAC transcription factor (TF) ATAF2 (ANAC081) and the core circadian clock regulator CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) both suppress the expression ofBAS1andSOB7via direct promoter binding. Additionally, BRs cause feedback suppression onATAF2expression. Here, we report that two ATAF‐subgroup TFs, ANAC102 and ATAF1 (ANAC002), also contribute to the transcriptional suppression ofBAS1andSOB7.ANAC102andATAF1gene‐knockout mutants exhibit elevated expression of bothBAS1andSOB7, expanded tissue‐level accumulation of their protein products and reduced hypocotyl growth in response to exogenous BR treatments. Similar toATAF2, bothANAC102andATAF1are transcriptionally suppressed by BRs and white light. NeitherBAS1norSOB7expression is further elevated inATAFdouble or triple mutants, suggesting that the suppression effect of these three ATAFs is not additive. In addition,ATAFsingle, double, and triple mutants have similar levels of BR responsiveness with regard to hypocotyl elongation. ATAF2, ANAC102, ATAF1, and CCA1 physically interact with itself and each other, suggesting that they may coordinately suppressBAS1andSOB7expression via protein–protein interactions. Despite the absence of CCA1‐binding elements in their promoters,ANAC102andATAF1have similar transcript circadian oscillation patterns as that ofCCA1, suggesting that these twoATAFgenes may be indirectly regulated by the circadian clock.

    

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2. LncRNA FLAIL affects alternative splicing and represses flowering in Arabidopsis

   https://doi.org/10.15252/embj.2022110921  

   Jin, Yu ; Ivanov, Maxim ; Dittrich, Anna Nelson ; Nelson, Andrew DL ; Marquardt, Sebastian ( April 2023 , The EMBO Journal)

   How the noncoding genome affects cellular functions is a key biological question. A particular challenge is to distinguish the effects of noncoding DNA elements from long noncoding RNAs (lncRNAs) that coincide at the same loci. Here, we identified the flowering‐associated intergenic lncRNA (FLAIL) inArabidopsisthrough early floweringflailmutants. Expression ofFLAILRNA from a different chromosomal location in combination with strand‐specific RNA knockdown characterizedFLAILas a trans‐acting RNA molecule.FLAILdirectly binds to differentially expressed target genes that control flowering via RNA–DNA interactions through conserved sequence motifs.FLAILinteracts with protein and RNA components of the spliceosome to affect target mRNA expression through co‐transcriptional alternative splicing (AS) and linked chromatin regulation. In the absence ofFLAIL, splicing defects at the direct FLAIL target flowering gene LACCASE 8 (LAC8) correlated with reduced mRNA expression. Double mutant analyses support a model whereFLAIL‐mediated splicing of LAC8 promotes its mRNA expression and represses flowering. Our study suggests lncRNAs as accessory components of the spliceosome that regulate AS and gene expression to impact organismal development.

    

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3. slim shady is a novel allele of PHYTOCHROME B present in the T‐DNA line SALK_015201

   https://doi.org/10.1002/pld3.326  

   Dash, Linkan ; McEwan, Robert E. ; Montes, Christian ; Mejia, Ludvin ; Walley, Justin W. ; Dilkes, Brian P. ; Kelley, Dior R. ( June 2021 , Plant Direct)

   Auxin is a hormone that is required for hypocotyl elongation during seedling development. In response to auxin, rapid changes in transcript and protein abundance occur in hypocotyls, and some auxin responsive gene expression is linked to hypocotyl growth. To functionally validate proteomic studies, a reverse genetics screen was performed on mutants in auxin‐regulated proteins to identify novel regulators of plant growth. This uncovered a long hypocotyl mutant, which we calledslim shady, in an annotated insertion line inIMMUNOREGULATORY RNA‐BINDING PROTEIN(IRR). Overexpression of theIRRgene failed to rescue theslim shadyphenotype and characterization of a second T‐DNA allele of IRR found that it had a wild‐type (WT) hypocotyl length. Theslim shadymutant has an elevated expression of numerous genes associated with the brassinosteroid‐auxin‐phytochrome (BAP) regulatory module compared to WT, including transcription factors that regulate brassinosteroid, auxin, and phytochrome pathways. Additionally,slim shadyseedlings fail to exhibit a strong transcriptional response to auxin. Using whole genome sequence data and genetic complementation analysis with SALK_015201C, we determined that a novel single nucleotide polymorphism inPHYTOCHROME Bwas responsible for theslim shadyphenotype. This is predicted to induce a frameshift and premature stop codon at leucine 1125, within the histidine kinase‐related domain of the carboxy terminus of PHYB, which is required for phytochrome signaling and function. Genetic complementation analyses withphyb‐9confirmed thatslim shadyis a mutant allele ofPHYB. This study advances our understanding of the molecular mechanisms in seedling development, by furthering our understanding of how light signaling is linked to auxin‐dependent cell elongation. Furthermore, this study highlights the importance of confirming the genetic identity of research material before attributing phenotypes to known mutations sourced from T‐DNA stocks.

    

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4. Transcriptome and phenotyping analyses support a role for chloroplast sigma factor 2 in red‐light‐dependent regulation of growth, stress, and photosynthesis

   https://doi.org/10.1002/pld3.43  

   Oh, Sookyung ; Strand, Deserah D. ; Kramer, David M. ; Chen, Jin ; Montgomery, Beronda L. ( February 2018 , Plant Direct)

   Sigma factor (SIG) proteins contribute to promoter specificity of the plastid‐encodedRNApolymerase during chloroplast genome transcription. All six members of theSIGfamily, that is,SIG1–SIG6, are nuclear‐encoded proteins targeted to chloroplasts. Sigma factor 2 (SIG2) is a phytochrome‐regulated protein important for stoichiometric control of the expression of plastid‐ and nuclear‐encoded genes that impact plastid development and plant growth and development. AmongSIGfactors,SIG2 is required not only for transcription of chloroplast genes (i.e., anterograde signaling), but also impacts nuclear‐encoded, photosynthesis‐related, and light signaling‐related genes (i.e., retrograde signaling) in response to plastid functional status. AlthoughSIG2 is involved in photomorphogenesis in Arabidopsis, the molecular bases for its role in light signaling that impacts photomorphogenesis and aspects of photosynthesis have only recently begun to be investigated. Previously, we reported thatSIG2 is necessary for phytochrome‐mediated photomorphogenesis specifically under red (R) and far‐red light, thereby suggesting a link between phytochromes and nuclear‐encodedSIG2 in light signaling. To explore transcriptional roles ofSIG2 in R‐dependent growth and development, we performedRNAsequencing analysis to compare gene expression insig2‐2mutant and Col‐0 wild‐type seedlings at two developmental stages (1‐ and 7‐day). We identified a subset of misregulated genes involved in growth, hormonal cross talk, stress responses, and photosynthesis. To investigate the functional relevance of these gene expression analyses, we performed several comparative phenotyping tests. In these analyses, strongsig2mutants showed insensitivity to bioactiveGA₃, high intracellular levels of hydrogen peroxide (H₂O₂) indicative of a stress response, and specific defects in photosynthesis, including elevated levels of cyclic electron flow (CEF) and nonphotochemical quenching (NPQ). We demonstrated thatSIG2 regulates a broader range of physiological responses at the molecular level than previously reported, with specific roles in red‐light‐mediated photomorphogenesis.

    

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5. Oncogenic KRAS alters splicing factor phosphorylation and alternative splicing in lung cancer

   https://doi.org/10.1186/s12885-022-10311-1  

   Lo, April ; McSharry, Maria ; Berger, Alice H. ( December 2022 , BMC Cancer)

   Alternative RNA splicing is widely dysregulated in cancers including lung adenocarcinoma, where aberrant splicing events are frequently caused by somatic splice site mutations or somatic mutations of splicing factor genes. However, the majority of mis-splicing in cancers is unexplained by these known mechanisms. We hypothesize that the aberrant Ras signaling characteristic of lung cancers plays a role in promoting the alternative splicing observed in tumors.

   We recently performed transcriptome and proteome profiling of human lung epithelial cells ectopically expressing oncogenic KRAS and another cancer-associated Ras GTPase, RIT1. Unbiased analysis of phosphoproteome data identified altered splicing factor phosphorylation in KRAS-mutant cells, so we performed differential alternative splicing analysis using rMATS to identify significantly altered isoforms in lung epithelial cells. To determine whether these isoforms were uniquely regulated by KRAS, we performed a large-scale splicing screen in which we generated over 300 unique RNA sequencing profiles of isogenic A549 lung adenocarcinoma cells ectopically expressing 75 different wild-type or variant alleles across 28 genes implicated in lung cancer.

   Mass spectrometry data showed widespread downregulation of splicing factor phosphorylation in lung epithelial cells expressing mutant KRAS compared to cells expressing wild-type KRAS. We observed alternative splicing in the same cells, with 2196 and 2416 skipped exon events in KRAS^G12Vand KRAS^Q61Hcells, respectively, 997 of which were shared (p < 0.001 by hypergeometric test). In the high-throughput splicing screen, mutant KRAS induced the greatest number of differential alternative splicing events, second only to the RNA binding protein RBM45 and its variant RBM45^M126I. We identified ten high confidence cassette exon events across multiple KRAS variants and cell lines. These included differential splicing of the Myc Associated Zinc Finger (MAZ). As MAZ regulates expression of KRAS, this splice variant may be a mechanism for the cell to modulate wild-type KRAS levels in the presence of oncogenic KRAS.

   Proteomic and transcriptomic profiling of lung epithelial cells uncovered splicing factor phosphorylation and mRNA splicing events regulated by oncogenic KRAS. These data suggest that in addition to widespread transcriptional changes, the Ras signaling pathway can promote post-transcriptional splicing changes that may contribute to oncogenic processes.

    

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