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. 2010 Jun 21;5(6):e11173.
doi: 10.1371/journal.pone.0011173.

Alu sequences in undifferentiated human embryonic stem cells display high levels of A-to-I RNA editing

Sivan Osenberg  1 , Nurit Paz Yaacov, Michal Safran, Sharon Moshkovitz, Ronit Shtrichman, Ofra Sherf, Jasmine Jacob-Hirsch, Gilmor Keshet, Ninette Amariglio, Joseph Itskovitz-Eldor, Gideon Rechavi
Affiliations

Alu sequences in undifferentiated human embryonic stem cells display high levels of A-to-I RNA editing

Sivan Osenberg et al. PLoS One. .

Abstract

Adenosine to Inosine (A-to-I) RNA editing is a site-specific modification of RNA transcripts, catalyzed by members of the ADAR (Adenosine Deaminase Acting on RNA) protein family. RNA editing occurs in human RNA in thousands of different sites. Some of the sites are located in protein-coding regions but the majority is found in non-coding regions, such as 3'UTRs, 5'UTRs and introns - mainly in Alu elements. While editing is found in all tissues, the highest levels of editing are found in the brain. It was shown that editing levels within protein-coding regions are increased during embryogenesis and after birth and that RNA editing is crucial for organism viability as well as for normal development. In this study we characterized the A-to-I RNA editing phenomenon during neuronal and spontaneous differentiation of human embryonic stem cells (hESCs). We identified high editing levels of Alu repetitive elements in hESCs and demonstrated a global decrease in editing levels of non-coding Alu sites when hESCs are differentiating, particularly into the neural lineage. Using RNA interference, we showed that the elevated editing levels of Alu elements in undifferentiated hESCs are highly dependent on ADAR1. DNA microarray analysis showed that ADAR1 knockdown has a global effect on gene expression in hESCs and leads to a significant increase in RNA expression levels of genes involved in differentiation and development processes, including neurogenesis. Taken together, we speculate that A-to-I editing of Alu sequences plays a role in the regulation of hESC early differentiation decisions.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. RNA editing within protein- coding regions during spontaneous and neuronal differentiation of hESCs.
H9.2 hESCs were differentiated spontaneously or to highly enriched neuronal culture by EBs derivation or by using all-trans retinoic acid, respectively. The editing levels of BLCAP, CYFIP2, and FLNA mRNAs at sites: Y/C, K/E and Q/R, respectively, were measured by the SEQUENOME MassArray technology. The editing levels of 5HT2CR mRNA at sites: A, B, C, D and E and the editing level of GluR2 at site Q/R were measured by direct sequencing. Absence of bars means that editing is under the detection level.
Figure 2
Figure 2. RNA editing levels within Alu sequences decrease during neuronal and spontaneous differentiation of hESCs.
The editing levels within Alu sequences of eight genes during the differentiation of H9.2 hESCs were measured. (A) RNA editing levels of C4orf29, F11R and THRAP1 Alu sequences were measured by direct sequencing. C4orf29 editing levels represent the average of 10 sites and F11R editing levels represent the average of 11 sites. Editing levels within Alu sequences of: RBBP9, FANCC, MDM4, BRCA1 and CARD11 were measured by the SEQUENOME MassArray technology in specific discrete sites. No editing was detected in the Alu of THRAP1 (B, C) A detailed representation of editing levels of the Alu elements of C4orf29 and F11R in the undifferentiated hESCs, the neuronal culture and the EBs. Specific localization of each editing site is given in table S7.
Figure 3
Figure 3. ADAR expression levels increase during neuronal and spontaneous differentiation of hESCs.
(A) The relative expression levels of ADAR mRNAs during H9.2 hESCs differentiations. The analysis was performed by qRT-PCR. Normalization was performed by GAPDH. (B) Western blot analysis of ADAR proteins expression levels during H9.2 hESCs differentiations. lane1: Undifferentiated hESCs, lane2: Neuronal culture, lane3: EBs, lane4: MEFs. ACTB -beta Actin.
Figure 4
Figure 4. RNA editing levels of Alu sequences are highly dependant on ADAR1 in hESCs.
H9.2 hESCs were grown under feeder-free conditions and transfected with ADAR1 siRNA or with non-_target negative control siRNA. 44 h and 72 h after tranfection, total RNA and protein were derived for analysis. (A) The relative mRNAs expression levels of ADAR1 p110 and ADAR1 p150 from the siRNAs transfected hESCs. Relative expression levels were measured by qRT-PCR. Normalization was done using GAPDH. (B) Western blot analysis of ADAR1 p110 and ADAR1 p150 proteins after the siRNA transfections. Lane 1 and lane 2 refer to ADAR1-siRNA and non-_target control siRNA transfected cultures after 44 h, respectively. Lane 3 and lane 4 refer to ADAR1-siRNA and control siRNA transfected cultures after 72 h, respectively. (C) The editing levels of BLCAP (Y/C) site and Alu sequences of six other genes in ADAR1 knockdown versus control hESCs. RNA editing levels of C4orf29 and F11R Alu sequences were measured by direct sequencing. C4orf29-Alu editing levels represent the average of 10 sites and F11R-Alu editing levels represent the average of 11 sites. Editing levels of the BRCA1, FANCC, RBBP9 and MDM4 tanscripts were measured by primer extension followed by mass spectrometry using the Sequenom MassArray in specific discrete sites. (D, E) A detailed representation of editing levels in the Alu sequences of C4orf29 and F11R in ADAR1 knockdown versus control hESCs which were measured by direct sequencing. Specific localization of each editing site is given in Table S7.
Figure 5
Figure 5. A global affect of ADAR1 silencing on gene expression in hESCs.
44 h after transfections, RNA were derived from hESCs treated with ADAR1-siRNA or with non-_target siRNA negative control and analyzed by oligonucleotide Microarray. Annotated genes whose expression level was changed at least at 1.5 fold as a result of ADAR1 silencing were categorized into functional groups. Prominent functional groups of genes which were overrepresented in the upregulated and/or downregulated genes are shown. One star indicates p-value<0.05. Two stars indicate p-value<0.001.
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