Papers by Christina Godfried Sie
The selective deamination of adenosines (A) to inosines (I) in messenger RNAs (mRNAs) can alter t... more The selective deamination of adenosines (A) to inosines (I) in messenger RNAs (mRNAs) can alter the encoded protein's amino acid sequence, with often critical consequences on protein stability, localization, and/or function. Insulin-like growth factor-binding protein 7 (IGFBP7) supports cell-adhesion and stimulates fibroblast proliferation with IGF and insulin. It exists in both proteolytically processed and unprocessed forms with altered cell-extracellular matrix interactions. Here we show that editing of IGFBP7 transcripts impacts the protein's susceptibility to proteolytic cleavage, thus providing a means for a cell to modulate its functionality through A-to-I RNA editing.
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A-to-I RNA editing results in the conversion of single adenosines into inosines, which alters cod... more A-to-I RNA editing results in the conversion of single adenosines into inosines, which alters coding and non-coding sequences in RNA molecules, increasing the complexity of the transcriptome. This modification is vital in a number of brain-specific coding transcripts, where the introduced alternative amino acids impact protein function substantially. Indeed, deviations from normal editing levels have been detected in tissues from individuals affected by neurological diseases and cancer, underscoring the importance of correct and regulated editing. Since the discovery of A-to-I RNA editing, considerable effort has been made to uncover additional editing targets and analyze the subsequent functional consequences for the recoded substrates. The effects of editing on non-coding RNAs (ncRNAs) such as microRNAs (miRNAs) or long ncRNAs are less well explored. ncRNAs act as regulators of gene expression through chromatin modification, imprinting, alternative splicing, and mRNA translation and stability. Editing has the potential to dynamically alter and diversify ncRNAs, thereby redirecting their functions. How editing intersects, interferes with, and modulates the roles of ncRNAs, possibly in response to external stimuli, therefore warrants a deeper look. This review discusses recent advances and new insights in the field.
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RNA editing by adenosine deamination, catalyzed by adenosine deaminases acting on RNA (ADAR), is ... more RNA editing by adenosine deamination, catalyzed by adenosine deaminases acting on RNA (ADAR), is a post-transcriptional modification that contributes to transcriptome and proteome diversity and is widespread in mammals. Here we administer a bioinformatics search strategy to the human and mouse genomes to explore the landscape of A-to-I RNA editing. In both organisms we find evidence for high excess of A/G-type discrepancies (inosine appears as a guanosine in cloned cDNA) at non-polymorphic, non-synonymous codon sites over other types of discrepancies, suggesting the existence of several thousand recoding editing sites in the human and mouse genomes. We experimentally validate recoding-type A-to-I RNA editing in a number of human genes with high scoring positions including the coatomer protein complex subunit alpha (COPA) as well as cyclin dependent kinase CDK13.
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Sie, CP., Maas, S.
A-to-I RNA editing can lead to recoding of pre-mRNAs with profound functional... more Sie, CP., Maas, S.
A-to-I RNA editing can lead to recoding of pre-mRNAs with profound functional consequences for the ensuing proteins. Here we show that complement component 1, q subcomponent-like 1 (C1QL1) undergoes RNA editing in vivo causing non-synonymous amino acid substitutions in human, mouse as well as zebrafish. The major editing site had previously been annotated as a single-nucleotide polymorphism in human, but our analysis reveals that post-transcriptional modification is the cause for the sequence variation. Remarkably, although editing of C1QL1 is conserved across vertebrate species, the predicted RNA secondary structure mediating editing involves different regions in zebrafish versus mammals.
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Single nucleotide polymorphisms (SNPs) are DNA sequence variations that can affect the expression... more Single nucleotide polymorphisms (SNPs) are DNA sequence variations that can affect the expression or function of genes. As a result, they may lead to phenotypic differences between individuals, such as susceptibility to disease, response to medications, and disease progression. Millions of SNPs have been mapped within the human genome providing a rich resource for genetic variation studies. Adenosine-to-inosine RNA editing also leads to the production of RNA and protein sequence variants, but it acts on the level of primary gene transcripts. Sequence variations due to RNA editing may be misannotated as SNPs when relying solely on expressed sequence data instead of genomic material. In this study, we screened the human SNP database for potential cases of A-to-I RNA editing that cause amino acid changes in the encoded protein. Our search strategy applies five molecular features to score candidate sites. It identifies all previously known cases of editing present in the SNP database and successfully uncovers novel, bona fide targets of adenosine deamination editing. Our approach sets the stage for effective and comprehensive genome-wide screens for A-to-I editing targets.
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Mutations in BRCA1 tumor suppressor account for ~45% of hereditary breast cancer and predispose i... more Mutations in BRCA1 tumor suppressor account for ~45% of hereditary breast cancer and predispose individuals to ovarian and prostate tumorigenesis. BRCA1-deficient cells contain numerous chromosome aberrations (duplications, translocations, inter-sister gaps) and defects in gene regulation. While BRCA1 binds numerous factors to engage in a wide variety of activities (ubiquitin ligase, DNA repair/damage signaling, chromatin remodeling, transcription activation), how BRCA1 mutations affect genetic instability at the molecular level remains unclear.
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The expansion of chondrocytes in automated bioreactors for clinical use requires that a relevant ... more The expansion of chondrocytes in automated bioreactors for clinical use requires that a relevant number of cells be generated, starting from variable initial seeding densities in one passage and using autologous serum. We investigated whether the growth factor combination transforming growth factor beta 1/fibroblast growth factor 2/platelet-derived growth factor BB (TFP), recently shown to enhance the proliferation capacity of human articular chondrocytes (HACs), allows the efficiency of chondrocyte use to be increased at different seeding densities and percentages of human serum (HS). HACs were seeded at 1,000, 5,000, and 10,000 cells/cm2 in medium containing 10% fetal bovine serum or 10,000 cells/cm2 with 1%, 5%, or 10%HS. The chondrogenic capacity of post-expanded HACs was then assessed in pellet cultures. Expansion with TFP allowed a sufficient number of HACs to be obtained in one passage even at the lowest seeding density and HS percentage and variability in cartilage-forming capacity of HACs expanded under the different conditions to be reduced. Instead, larger variations and insufficient yields were found in the absence of TFP. By allowing large numbers of cells to be obtained, starting from a wide range of initial seeding densities and HS percentages, the use of TFP may represent a viable solution for the efficient expansion of HACs and addresses constraints of automated clinical bioreactor systems.
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Talks by Christina Godfried Sie
Our lab employed a computational algorithm which is specifically designed to search for potential... more Our lab employed a computational algorithm which is specifically designed to search for potential A-to-I RNA editing targets in sequence databases and which ranks them according to the strength of ADAR-preferred molecular features. SerpinA3 emerged as top-scoring candidate with a predicted 90bp completely complementary double-stranded structure, which is formed by sequences contained in exon 3 and intron 3. Extensive editing is generally observed in mRNAs with double-stranded sequences of 50bp or more; however, analysis of the SerpinA3 exonic sequence by PCR amplification and Sanger sequencing did not reveal evidence of in vivo editing in the analyzed tissue samples. We are now investigating if cis-acting sequences may make this seemingly perfect target immune to editing, which may shed further light on how editing is regulated in the cell.
To this end, we are using a minigene construct that expresses the SerpinA3 gene from exon 3 to exon 4 in HeLa cells. The minigene was manipulated by site-directed mutagenesis in order to localize cis-acting sequence factors that contribute to the prevention of editing. In particular, we hypothesized that formation of the predicted secondary structure is thwarted by one or more of the following: partial LINE2 elements in the intron could potentially promote the formation of an alternative fold; splicing, though previously shown to be coordinated with editing, could remove the ECS before the predicted structure can form; and/or protein factors, such as splice enhancers, that bind to the RNA could impede folding of the two 90nt complementary sequences. These specific sequence elements were mutated and the corresponding transcripts from transfected HeLa cells were analyzed with respect to editing in the target sequence.
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Papers by Christina Godfried Sie
A-to-I RNA editing can lead to recoding of pre-mRNAs with profound functional consequences for the ensuing proteins. Here we show that complement component 1, q subcomponent-like 1 (C1QL1) undergoes RNA editing in vivo causing non-synonymous amino acid substitutions in human, mouse as well as zebrafish. The major editing site had previously been annotated as a single-nucleotide polymorphism in human, but our analysis reveals that post-transcriptional modification is the cause for the sequence variation. Remarkably, although editing of C1QL1 is conserved across vertebrate species, the predicted RNA secondary structure mediating editing involves different regions in zebrafish versus mammals.
Talks by Christina Godfried Sie
To this end, we are using a minigene construct that expresses the SerpinA3 gene from exon 3 to exon 4 in HeLa cells. The minigene was manipulated by site-directed mutagenesis in order to localize cis-acting sequence factors that contribute to the prevention of editing. In particular, we hypothesized that formation of the predicted secondary structure is thwarted by one or more of the following: partial LINE2 elements in the intron could potentially promote the formation of an alternative fold; splicing, though previously shown to be coordinated with editing, could remove the ECS before the predicted structure can form; and/or protein factors, such as splice enhancers, that bind to the RNA could impede folding of the two 90nt complementary sequences. These specific sequence elements were mutated and the corresponding transcripts from transfected HeLa cells were analyzed with respect to editing in the target sequence.
A-to-I RNA editing can lead to recoding of pre-mRNAs with profound functional consequences for the ensuing proteins. Here we show that complement component 1, q subcomponent-like 1 (C1QL1) undergoes RNA editing in vivo causing non-synonymous amino acid substitutions in human, mouse as well as zebrafish. The major editing site had previously been annotated as a single-nucleotide polymorphism in human, but our analysis reveals that post-transcriptional modification is the cause for the sequence variation. Remarkably, although editing of C1QL1 is conserved across vertebrate species, the predicted RNA secondary structure mediating editing involves different regions in zebrafish versus mammals.
To this end, we are using a minigene construct that expresses the SerpinA3 gene from exon 3 to exon 4 in HeLa cells. The minigene was manipulated by site-directed mutagenesis in order to localize cis-acting sequence factors that contribute to the prevention of editing. In particular, we hypothesized that formation of the predicted secondary structure is thwarted by one or more of the following: partial LINE2 elements in the intron could potentially promote the formation of an alternative fold; splicing, though previously shown to be coordinated with editing, could remove the ECS before the predicted structure can form; and/or protein factors, such as splice enhancers, that bind to the RNA could impede folding of the two 90nt complementary sequences. These specific sequence elements were mutated and the corresponding transcripts from transfected HeLa cells were analyzed with respect to editing in the target sequence.