Cloning, Characterization, and Overexpression in Arabidopsis to Determine the Function of SPL Genes from Woodland Strawberry (Fragaria Vesca)

2021 ◽  
Author(s):  
Yibo Bai ◽  
Morong Liang ◽  
Chuangju Ma ◽  
Zongming Cheng ◽  
Jinsong Xiong
Keyword(s):  
Untranslated Region ◽  
Transcriptional Activity ◽  
Biochemical Analysis ◽  
Activity Analysis ◽  
Fragaria Vesca ◽  
Mobility Shift ◽  
Woodland Strawberry ◽  
Squamosa Promoter Binding Protein ◽  
Spl Genes ◽  
Mobility Shift Assay

Abstract SQUAMOSA promoter binding protein-like (SPL) proteins is a class of plant specific transcription factors that play important roles during plant development. However, the majority of SPL genes in strawberry are functionally uncharacterized. In this study, three SPL genes, i.e. FvSPL1, FvSPL2, and FvSPL11 (FvSPL1/2/11), from woodland strawberry were cloned and characterized. Phylogenetic analysis with SPL genes from Arabidopsis, tomato and chrysanthemum indicated that FvSPL1/2/11 were clustered into the same group with those of miR156 target site located at the 3’-untranslated region (UTR). Further biochemical analysis indicated that FvSPL1 was exclusively localized in the nucleus. Electrophoretic mobility shift assay demonstrated FvSPL1 could specifically recognized the GTAC motif. Transcriptional activity analysis showed FvSPL1 was a transcriptional activator that could activate the expression of FvAP1 gene. Finally, all of the transgenic Arabidopsis that overexpression the three FvSPL genes were exhibited significantly early flowering phenotype. Taken together, our study indicated that FvSPL1/2/11 similar to their orthologs in Arabidopsis mainly functions in regulating plant flowering. These results enriched our understanding to the functions of SPL genes in strawberry and might be utilized for strawberry flowering time manipulation in the future.

Genome-wide identification and expression analysis of the SPL gene family in woodland strawberry Fragaria vesca

Genome ◽  
2018 ◽  
Vol 61 (9) ◽  
pp. 675-683 ◽  
Author(s):  
Jin-Song Xiong ◽  
Dan Zheng ◽  
Hong-Yu Zhu ◽  
Jian-Qiu Chen ◽  
Ran Na ◽  
...  
Keyword(s):  
Expression Analysis ◽  
Growth And Development ◽  
Expression Patterns ◽  
Systematic Research ◽  
Fragaria Vesca ◽  
Woodland Strawberry ◽  
Genome Wide ◽  
Squamosa Promoter Binding Protein ◽  
Spl Genes ◽  
Localization Analysis

SQUAMOSA promoter-binding protein-like (SPL) is a class of plant-specific transcription factors that play critical roles in regulating plant growth and development. However, little systematic research on SPL genes has been conducted in strawberry. In this study, 14 SPL genes were identified in the genome of woodland strawberry (Fragaria vesca), one of the model plants of the family Rosaceae. Chromosome localization analysis indicated that the 14 FvSPL genes were unevenly distributed on six chromosomes. Phylogenetic analysis indicated that the FvSPL proteins could be clustered into six groups (G1 to G6). Genes with similar structure were classified into the same group, implying their functional redundancy. In addition, nine out of the 14 FvSPL genes, belonging to G1, G2, and G5, were found to be the putative targets of FvmiR156 genes. Expression analysis indicated FvSPL genes exhibited highly diverse expression patterns in the tissues and organs examined. The transcript levels of most FvmiR156-targeted FvSPL genes in fruit were lower than those non-miR156-targeted genes. In addition, the expression of the FvmiR156-targeted FvSPL genes decreased during fruit ripening, whereas the expression of FvmiR156 genes increased in fruit during this process. The results provide a foundation for future functional analysis of FvSPL genes in strawberry growth and development.

scholarly journals Nucleolin stabilizes G-quadruplex structures folded by the LTR promoter and silences HIV-1 viral transcription

2015 ◽  
Vol 43 (18) ◽  
pp. 8884-8897 ◽  
Author(s):  
Elena Tosoni ◽  
Ilaria Frasson ◽  
Matteo Scalabrin ◽  
Rosalba Perrone ◽  
Elena Butovskaya ◽  
...  
Keyword(s):  
Transcriptional Activity ◽  
Cellular Protein ◽  
Spectrometric Analysis ◽  
Viral Transcription ◽  
Mobility Shift ◽  
Binding Preference ◽  
G Quadruplex ◽  
Mobility Shift Assay ◽  
Ltr Promoter ◽  
Hiv 1

Abstract Folding of the LTR promoter into dynamic G-quadruplex conformations has been shown to suppress its transcriptional activity in HIV-1. Here we sought to identify the proteins that control the folding of this region of proviral genome by inducing/stabilizing G-quadruplex structures. The implementation of electrophorethic mobility shift assay and pull-down experiments coupled with mass spectrometric analysis revealed that the cellular protein nucleolin is able to specifically recognize G-quadruplex structures present in the LTR promoter. Nucleolin recognized with high affinity and specificity the majority, but not all the possible G-quadruplexes folded by this sequence. In addition, it displayed greater binding preference towards DNA than RNA G-quadruplexes, thus indicating two levels of selectivity based on the sequence and nature of the target. The interaction translated into stabilization of the LTR G-quadruplexes and increased promoter silencing activity; in contrast, disruption of nucleolin binding in cells by both siRNAs and a nucleolin binding aptamer greatly increased LTR promoter activity. These data indicate that nucleolin possesses a specific and regulated activity toward the HIV-1 LTR promoter, which is mediated by G-quadruplexes. These observations provide new essential insights into viral transcription and a possible low mutagenic target for antiretroviral therapy.

U-rich sequence-binding proteins (URBPs) interacting with a 20-nucleotide U-rich sequence in the 3' untranslated region of c-fos mRNA may be involved in the first step of c-fos mRNA degradation

1992 ◽  
Vol 12 (7) ◽  
pp. 2931-2940
Author(s):  
Y You ◽  
C Y Chen ◽  
A B Shyu
Keyword(s):  
Untranslated Region ◽  
Binding Proteins ◽  
Critical Role ◽  
Point Mutations ◽  
Mrna Degradation ◽  
Binding Activity ◽  
Early Gene ◽  
Mobility Shift ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift

Rapid decay of the c-fos transcript plays a critical role in controlling transforming potential of the c-fos proto-oncogene. One of the mRNA instability determinants is a 75-nucleotide AU-rich element (ARE) present in the 3' untranslated region of the c-fos transcript. It appears to control two steps in the process of c-fos mRNA degradation: removal of the poly(A) tail, which does not require the AUUUA motifs, and subsequent degradation of deadenylated mRNA, which appears to be dependent on the AUUUA motifs. In this study, we report the identification of four U-rich sequence binding proteins (URBPs) that specifically interact with a 20-nucleotide U-rich sequence within the c-fos ARE. Gel mobility shift assay and competition experiments showed that these protein factors form three specific band-shifted complexes with the c-fos ARE. Binding activity of one of the protein factors, a 37-kDa protein, is significantly affected by serum induction and by pretreatment of cells with drugs known to stabilize many of the immediate-early gene mRNAs. Combining UV cross-linking with a new approach, designated sequential RNase digestion, we were able to better determine the molecular masses of these cellular proteins. The binding sites for the four proteins were all mapped to a 20-nucleotide U-rich sequence located at the 3' half of the c-fos ARE, which contains no AUUUA pentanucleotides but stretches of uridylate residues. Single U-to-A point mutations in each of the three AUUUA motifs within the c-fos ARE have little effect on formation of the mobility-shifted complexes. Our data indicate c-fos ARE-protein interaction involves recognition of U stretches rather than recognition of the AUUUA motifs. We propose that UTBP binding may be involved in the first step, removal of the Poly(A) tail, in the c-fos ARE-mediated decay pathway.

scholarly journals Regulation of lactase–phlorizin hydrolase gene expression by the caudal-related homoeodomain protein Cdx-2

1997 ◽  
Vol 322 (3) ◽  
pp. 833-838 ◽  
Author(s):  
Jesper T. TROELSEN ◽  
Cathy MITCHELMORE ◽  
Nikolaj SPODSBERG ◽  
Anette M. JENSEN ◽  
Ove NORÉN ◽  
...  
Keyword(s):  
Gene Expression ◽  
Small Intestine ◽  
Binding Site ◽  
Electrophoretic Mobility Shift Assay ◽  
Gene Transcription ◽  
Transcriptional Activity ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Cis Element ◽  
Mobility Shift Assay

Lactase–phlorizin hydrolase is exclusively expressed in the small intestine and is often used as a marker for the differentiation of enterocytes. The cis-element CE-LPH1 found in the lactase–phlorizin hydrolase promoter has previously been shown to bind an intestinal-specific nuclear factor. By electrophoretic mobility-shift assay it was shown that the factor Cdx-2 (a homoeodomain-protein related to caudal) binds to a TTTAC sequence in the CE-LPH1. Furthermore it was demonstrated that Cdx-2 is able to activate reporter gene transcription by binding to CE-LPH1. A mutation in CE-LPH1, which does not affect Cdx-2 binding, results in a higher transcriptional activity, indicating that the CE-LPH1 site contains other binding site(s) in addition to the Cdx-2-binding site.

scholarly journals MSY2 and MSY4 Bind a Conserved Sequence in the 3′ Untranslated Region of Protamine 1 mRNA In Vitro and In Vivo

2001 ◽  
Vol 21 (20) ◽  
pp. 7010-7019 ◽  
Author(s):  
Flaviano Giorgini ◽  
Holly G. Davies ◽  
Robert E. Braun
Keyword(s):  
Untranslated Region ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Binding Activity ◽  
Mobility Shift ◽  
Conserved Sequence ◽  
Mobility Shift Assay ◽  
Protamine 1

ABSTRACT Y-box proteins are major constituents of ribonucleoprotein particles (RNPs) which contain translationally silent mRNAs in gametic cells. We have recently shown that a sequence-specific RNA binding activity present in spermatogenic cells contains the two Y-box proteins MSY2 and MSY4. We show here that MSY2 and MSY4 bind a sequence, 5′-UCCAUCA-3′, present in the 3′ untranslated region of the translationally repressed protamine 1 (Prm1) mRNA. Using pre- and post-RNase T1-digested substrate RNAs, it was determined that MSY2 and MSY4 can bind an RNA of eight nucleotides containing the MSY2 and MSY4 binding site. Single nucleotide mutations in the sequence eliminated the binding of MSY2 and MSY4 in an electrophoretic mobility shift assay, and the resulting mutants failed to compete for binding in a competition assay. A consensus site of UACCACAUCCACU(subscripts indicate nucleotides which do not disrupt YRS binding by MSY2 and MSY4), denoted the Y-box recognition site (YRS), was defined from this mutational analysis. These mutations in the YRS were further characterized in vivo using a novel application of the yeast three-hybrid system. Experiments with transgenic mice show that disruption of the YRS in vivo relieves Prm1-like repression of a reporter gene. The conservation of the RNA binding motifs among Y-box protein family members raises the possibility that other Y-box proteins may have previously unrecognized sequence-specific RNA binding activities.

scholarly journals Molecular Cloning and Characterization of SQUAMOSA-Promoter Binding Protein-Like Gene FvSPL10 from Woodland Strawberry (Fragaria vesca)

Plants ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 342 ◽  
Author(s):  
Jinsong Xiong ◽  
Yibo Bai ◽  
Chuangju Ma ◽  
Hongyu Zhu ◽  
Dan Zheng ◽  
...  
Keyword(s):  
Plant Growth ◽  
Growth And Development ◽  
Binding Protein ◽  
Ectopic Expression ◽  
Plant Growth And Development ◽  
Woodland Strawberry ◽  
Squamosa Promoter Binding Protein ◽  
Spl Genes

SQUAMOSA-promoter binding protein-like (SPL) proteins are plant-specific transcript factors that play essential roles in plant growth and development. Although many SPL genes have been well characterized in model plants like Arabidopsis, rice and tomato, the functions of SPLs in strawberry are still largely elusive. In the present study, we cloned and characterized FvSPL10, the ortholog of AtSPL9, from woodland strawberry. Subcellular localization shows FvSPL10 localizes in the cell nucleus. The luciferase system assay indicates FvSPL10 is a transcriptional activator, and both in vitro and in vivo assays indicate FvSPL10 could bind to the promoter of FvAP1 and activate its expression. Ectopic expression of FvSPL10 in Arabidopsis promotes early flowering and increases organs size. These results demonstrate the multiple regulatory roles of FvSPL10 in plant growth and development and lay a foundation for investigating the biological functions of FvSPL10 in strawberry.

scholarly journals U-rich sequence-binding proteins (URBPs) interacting with a 20-nucleotide U-rich sequence in the 3' untranslated region of c-fos mRNA may be involved in the first step of c-fos mRNA degradation.

1992 ◽  
Vol 12 (7) ◽  
pp. 2931-2940 ◽  
Author(s):  
Y You ◽  
C Y Chen ◽  
A B Shyu
Keyword(s):  
Untranslated Region ◽  
Binding Proteins ◽  
Critical Role ◽  
Point Mutations ◽  
Mrna Degradation ◽  
Binding Activity ◽  
Early Gene ◽  
Mobility Shift ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift

Rapid decay of the c-fos transcript plays a critical role in controlling transforming potential of the c-fos proto-oncogene. One of the mRNA instability determinants is a 75-nucleotide AU-rich element (ARE) present in the 3' untranslated region of the c-fos transcript. It appears to control two steps in the process of c-fos mRNA degradation: removal of the poly(A) tail, which does not require the AUUUA motifs, and subsequent degradation of deadenylated mRNA, which appears to be dependent on the AUUUA motifs. In this study, we report the identification of four U-rich sequence binding proteins (URBPs) that specifically interact with a 20-nucleotide U-rich sequence within the c-fos ARE. Gel mobility shift assay and competition experiments showed that these protein factors form three specific band-shifted complexes with the c-fos ARE. Binding activity of one of the protein factors, a 37-kDa protein, is significantly affected by serum induction and by pretreatment of cells with drugs known to stabilize many of the immediate-early gene mRNAs. Combining UV cross-linking with a new approach, designated sequential RNase digestion, we were able to better determine the molecular masses of these cellular proteins. The binding sites for the four proteins were all mapped to a 20-nucleotide U-rich sequence located at the 3' half of the c-fos ARE, which contains no AUUUA pentanucleotides but stretches of uridylate residues. Single U-to-A point mutations in each of the three AUUUA motifs within the c-fos ARE have little effect on formation of the mobility-shifted complexes. Our data indicate c-fos ARE-protein interaction involves recognition of U stretches rather than recognition of the AUUUA motifs. We propose that UTBP binding may be involved in the first step, removal of the Poly(A) tail, in the c-fos ARE-mediated decay pathway.

scholarly journals BrTCP7 Transcription Factor Is Associated with MeJA-Promoted Leaf Senescence by Activating the Expression of BrOPR3 and BrRCCR

2019 ◽  
Vol 20 (16) ◽  
pp. 3963 ◽  
Author(s):  
Yan-mei Xu ◽  
Xian-mei Xiao ◽  
Ze-xiang Zeng ◽  
Xiao-li Tan ◽  
Zong-li Liu ◽  
...  
Keyword(s):  
Leaf Senescence ◽  
Transcriptional Activity ◽  
Nuclear Protein ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Transient Expression Assay ◽  
Catabolic Genes ◽  
Meja Treatment ◽  
Mobility Shift Assay ◽  
Chlorophyll Catabolite

The plant hormone jasmonic acid (JA) has been recognized as an important promoter of leaf senescence in plants. However, upstream transcription factors (TFs) that control JA biosynthesis during JA-promoted leaf senescence remain unknown. In this study, we report the possible involvement of a TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) TF BrTCP7 in methyl jasmonate (MeJA)-promoted leaf senescence in Chinese flowering cabbage. Exogenous MeJA treatment reduced maximum quantum yield (Fv/Fm) and total chlorophyll content, accompanied by the increased expression of senescence marker and chlorophyll catabolic genes, and accelerated leaf senescence. To further understand the transcriptional regulation of MeJA-promoted leaf senescence, a class I member of TCP TFs BrTCP7 was examined. BrTCP7 is a nuclear protein and possesses trans-activation ability through subcellular localization and transcriptional activity assays. A higher level of BrTCP7 transcript was detected in senescing leaves, and its expression was up-regulated by MeJA. The electrophoretic mobility shift assay and transient expression assay showed that BrTCP7 binds to the promoter regions of a JA biosynthetic gene BrOPR3 encoding OPDA reductase3 (OPR3) and a chlorophyll catabolic gene BrRCCR encoding red chlorophyll catabolite reductase (RCCR), activating their transcriptions. Taken together, these findings reveal that BrTCP7 is associated with MeJA-promoted leaf senescence at least partly by activating JA biosynthesis and chlorophyll catabolism, thus expanding our knowledge of the transcriptional mechanism of JA-mediated leaf senescence.

scholarly journals A GG Nucleotide Sequence of the 3′ Untranslated Region of Amyloid Precursor Protein mRNA Plays a Key Role in the Regulation of Translation and the Binding of Proteins

2000 ◽  
Vol 20 (13) ◽  
pp. 4572-4579 ◽  
Author(s):  
E. G. Mbongolo Mbella ◽  
S. Bertrand ◽  
G. Huez ◽  
J.-N. Octave
Keyword(s):  
Amyloid Precursor Protein ◽  
Translational Control ◽  
Cho Cells ◽  
Untranslated Region ◽  
Specific Binding ◽  
Alternative Polyadenylation ◽  
Mrna Translation ◽  
Precursor Protein ◽  
Mobility Shift ◽  
Mobility Shift Assay

ABSTRACT The alternative polyadenylation of the mRNA encoding the amyloid precursor protein (APP) involved in Alzheimer's disease generates two molecules, with the first of these containing 258 additional nucleotides in the 3′ untranslated region (3′UTR). We have previously shown that these 258 nucleotides increase the translation of APP mRNA injected in Xenopus oocytes (5). Here, we demonstrate that this mechanism occurs in CHO cells as well. We also present evidence that the 3′UTR containing 8 nucleotides more than the short 3′UTR allows the recovery of an efficiency of translation similar to that of the long 3′UTR. Moreover, the two guanine residues located at the 3′ ends of these 8 nucleotides play a key role in the translational control. Using gel retardation mobility shift assay, we show that proteins from Xenopus oocytes, CHO cells, and human brain specifically bind to the short 3′UTR but not to the long one. The two guanine residues involved in the translational control inhibit this specific binding by 65%. These results indicate that there is a correlation between the binding of proteins to the 3′UTR of APP mRNA and the efficiency of mRNA translation, and that a GG motif controls both binding of proteins and translation.

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