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. 2008 Jan 23;3(1):e1486.
doi: 10.1371/journal.pone.0001486.

A novel RNA transcript with antiapoptotic function is silenced in fragile X syndrome

Ahmad M Khalil  1 , Mohammad Ali Faghihi, Farzaneh Modarresi, Shaun P Brothers, Claes Wahlestedt
Affiliations

A novel RNA transcript with antiapoptotic function is silenced in fragile X syndrome

Ahmad M Khalil et al. PLoS One. .

Abstract

Several genome-wide transcriptomics efforts have shown that a large percentage of the mammalian genome is transcribed into RNAs, however, only a small percentage (1-2%) of these RNAs is translated into proteins. Currently there is an intense interest in characterizing the function of the different classes of noncoding RNAs and their relevance to human disease. Using genomic approaches we discovered FMR4, a primate-specific noncoding RNA transcript (2.4 kb) that resides upstream and likely shares a bidirectional promoter with FMR1. FMR4 is a product of RNA polymerase II and has a similar half-life to FMR1. The CGG expansion in the 5' UTR of FMR1 appears to affect transcription in both directions as we found FMR4, similar to FMR1, to be silenced in fragile X patients and up-regulated in premutation carriers. Knockdown of FMR4 by several siRNAs did not affect FMR1 expression, nor vice versa, suggesting that FMR4 is not a direct regulatory transcript for FMR1. However, FMR4 markedly affected human cell proliferation in vitro; siRNAs knockdown of FMR4 resulted in alterations in the cell cycle and increased apoptosis, while the overexpression of FMR4 caused an increase in cell proliferation. Collectively, our results demonstrate an antiapoptotic function of FMR4 and provide evidence that a well-studied genomic locus can show unexpected functional complexity. It cannot be excluded that altered FMR4 expression might contribute to aspects of the clinical presentation of fragile X syndrome and/or related disorders.

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Conflict of interest statement

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

Figures

Figure 1
Figure 1. Identification and sequence analysis of FMR4.
(A) Schematic showing known genes in Xq27.3-28 including the newly identified FMR4. FMR4 is transcribed upstream of FMR1 and in the opposite direction. (B) Sequence of FMR4 obtained by rapid amplification of cDNA ends (RACE).
Figure 2
Figure 2. Expression analysis of FMR4.
(A) RT-PCR analysis of FMR4 and FMR1 in seven different human fetal tissues (week 12), RNA from each tissue was pooled from at least three fetuses (GBiosciences). The RNA expression of FMR1 and FMR4 were normalized to whole embryo (set as 100%). Both transcripts are expressed in all the tissues tested with notably high expression of FMR4 in the kidney and heart. (B) RNA was extracted from six postmortem human adult brains from three different regions, thereafter; cDNA synthesis followed by RT-PCR was performed on all samples to measure the relative quantities of FMR1 and FMR4. Both FMR1 and FMR4 are highly expressed in all the human brain regions tested. (C) RT-PCR analysis of FMR4 and FMR1 in several regions of two monkeys brains. The RNA expression of FMR4 and FMR1 were normalized to the insula (set as 100%).
Figure 3
Figure 3. FMR4 is silenced in fragile X syndrome.
(A) RNA from four normal, four premutation and four full mutation FXS patients isolated from untransformed leucocytes (kindly provided by Flora Tassone and Paul Hagerman, UC Davis) was reverse transcribed using random hexamers. Quantitative RT-PCR analysis revealed that FMR4, similar to FMR1, is up-regulated in pre-mutation carriers and shut down in full mutation fragile X patients (P<0.0001). (B) RNA from untransformed leucocytes were reversed transcribed and the cDNA was used for PCR analysis. FMR4 is expressed in normal and premutation carriers but no bands were observed in the full mutation fragile X patients (35 cycles). FMR1 bands were observed in normal, premutation, and one of the full mutation patients (35 cycles). To account for any possible DNA contamination, no reverse transcriptase control for all samples were used in the PCR (lanes next to bands are all negative indicating no DNA contamination was present). Error bars: s.d.
Figure 4
Figure 4. FMR4 has a similar half-life to FMR1.
HEK-293T cells were treated with α-amanitin (blocks RNA polymerase II) and the levels of FMR4 and FMR1 were measured by RT-PCR at 0, 6, 12 and 24 hours post treatment. Both FMR4 and FMR1 have similar half-lives. These experiments also further confirm that FMR4 is a product of RNA polymerase II.
Figure 5
Figure 5. No direct cross-regulation between FMR1 and FMR4.
(A) We used three distinct siRNAs against FMR1 to transfect HEK-293T cells. Two out of the three siRNAs resulted in a significant knockdown of FMR1 (80%), but did not affect FMR4 RNA levels. (B) We used three distinct siRNAs against FMR4 to transfect HEK-293T cells. All three siRNAs resulted in a significant knockdown of FMR4 but did not affect FMR1 RNA levels. (C) Significant knockdown of FMR4 via siRNA C did not result in a change in FMR1 RNA levels at any of the time points tested (24, 48, 72, or 144 hours post transfection). (D) The entire sequence of FMR4 was cloned into a pcDNA3.1 vector with a CMV promoter. The pcDNA3.1 vector containing the FMR4 sequence and the original pcDNA3.1 (without the FMR4 insert) were transfected in HEK-293T cells. At 72 hours post transfection, RNA was isolated and reversed transcribed and used for RT-PCR analysis. There is a highly significant increase in the FMR4 RNA levels but no effect on FMR1 RNA. Error bars: s.d.
Figure 6
Figure 6. FMR4 affects proliferation in human cells.
(A) Cell proliferation assay showing that the knockdown of FMR4 via three distinct siRNAs in HEK-293T cells, but not knockdown of FMR1, resulted in decrease in cell proliferation in comparison to cells which are treated with a negative control siRNA. Cell proliferation was measured based on luciferase activity in these cells at 72 hours post siRNA transfection. (B) Cell proliferation assay showing that the knockdown of FMR4 via three distinct siRNAs in HeLa cells resulted in decrease in cell proliferation in comparison to cells which are treated with a negative control siRNA (P<0.0001). (C–D) In both HEK-293T and HeLa cells, overexpression of FMR4 resulted in an increase in cell proliferation in comparison to cells treated with a control vector. Error bars: s.d.
Figure 7
Figure 7. The effect of FMR4 on cell proliferation is not observed in non-primates.
Since FMR4 is a primate-specific transcript we examined its effect on cell proliferation in non-primates using mouse N2A cells. We examined both the siRNA knockdown of FMR4 (as a negative control experiment) and the over-expression of FMR4 on cell proliferation in N2A cells. (A) Mouse N2A cells were transfected with FMR4 siRNA C and a control siRNA. Simultaneously, cells were transfected with pGL3 (luciferase) vector. At 72 hours post transfection luciferase activity was measured and data are graphed as a percentage of control siRNA. (B) Mouse N2A cells were transfected with FMR4 over-expression vector and a control vector (no FMR4 insert). Simultaneously, cells were transfected with pGL3 (luciferase) vector. At 72 hours post transfection luciferase activity was measured and data are graphed as a percentage of control vector. Unlike human cells which show an increase in cell proliferation when transfected with the FMR4 vector, mouse N2A cells did not show any change in proliferation.
Figure 8
Figure 8. FMR4 has an antiapoptotic function in human cells.
(A) Cell cycle analysis of control cells (red) and cells treated with two different siRNAs against FMR4 (green and blue) shows that knockdown of FMR4 resulted in a highly significant increase in the number of cells in Sub-G1 and a modest but significant decrease in the number of cells in the S phase suggesting a possible role in apoptosis. (B) Microscope images of cells (DAPI stained) treated with a control siRNA and cells treated with FMR4 siRNA for 72 hours prior to a TUNEL assay. A significant number of cells are undergoing apoptosis (FITC) in the FMR4 siRNA treated cells in comparison to the control siRNA treated cells. (C) Quantification of cells following a TUNEL assay indicated that there is at least a two-fold change in the number of cells undergoing apoptosis in the FMR4 siRNA treated cells in comparison to the control siRNA treated cells. Error bars: s.d.
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