RNA-Seq of human neurons derived from iPS cells reveals candidate long non-coding RNAs involved in neurogenesis and neuropsychiatric disorders - PubMed (original) (raw)

RNA-Seq of human neurons derived from iPS cells reveals candidate long non-coding RNAs involved in neurogenesis and neuropsychiatric disorders

Mingyan Lin et al. PLoS One. 2011.

Abstract

Genome-wide expression analysis using next generation sequencing (RNA-Seq) provides an opportunity for in-depth molecular profiling of fundamental biological processes, such as cellular differentiation and malignant transformation. Differentiating human neurons derived from induced pluripotent stem cells (iPSCs) provide an ideal system for RNA-Seq since defective neurogenesis caused by abnormalities in transcription factors, DNA methylation, and chromatin modifiers lie at the heart of some neuropsychiatric disorders. As a preliminary step towards applying next generation sequencing using neurons derived from patient-specific iPSCs, we have carried out an RNA-Seq analysis on control human neurons. Dramatic changes in the expression of coding genes, long non-coding RNAs (lncRNAs), pseudogenes, and splice isoforms were seen during the transition from pluripotent stem cells to early differentiating neurons. A number of genes that undergo radical changes in expression during this transition include candidates for schizophrenia (SZ), bipolar disorder (BD) and autism spectrum disorders (ASD) that function as transcription factors and chromatin modifiers, such as POU3F2 and ZNF804A, and genes coding for cell adhesion proteins implicated in these conditions including NRXN1 and NLGN1. In addition, a number of novel lncRNAs were found to undergo dramatic changes in expression, one of which is HOTAIRM1, a regulator of several HOXA genes during myelopoiesis. The increase we observed in differentiating neurons suggests a role in neurogenesis as well. Finally, several lncRNAs that map near SNPs associated with SZ in genome wide association studies also increase during neuronal differentiation, suggesting that these novel transcripts may be abnormally regulated in a subgroup of patients.

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Conflict of interest statement

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

Figures

Figure 1

Figure 1. Comparison of fold-changes (log2-transformed) of gene expression between iPSCs and day 10 neurons as measured by RNA-Seq (x-axis) and Affymetrix microarray (y-axis).

Figure 2

Figure 2. Validation by RT-PCR and qPCR in unamplified RNAs.

Panel A, top band in each of the 4 sets is a beta-actin control; bottom band, specific transcripts. Lane 1, iPSCs; lane 2, day 14 neurons; lane 3, day 27 neurons; lanes 4 and 5, fetal brain (9 and 20 weeks, respectively); lanes 6 and 7, adult prefrontal cortex; lane 8, no RT (negative control). Panel B, same as A, but no 9 week fetal sample (lane 4 is 20 week fetal brain and lanes 5 and 6 are adult prefrontal cortex. Panel C, qPCR for MIAT and CRNDE in iPSCs (white bar) and day 14 neurons (black bar). Relative changes in gene expression were calculated using the 2−ΔΔCt method with β2-microglobulin (β2M) as a reference gene. Mean increase in transcripts in neurons was statistically significant (p<0.05). Error bar is SD based on 3 or 4 replicates, each done in triplicate.

Figure 3

Figure 3. Splice variants in NRXN1 (panel A) and NRG1 (panel B).

Asterisk in A shows splicing across exons 19, 20 and 21 (exons 20 and 21 are only ∼2 kb apart and appear superimposed in figure). Asterisk in B covers exons 10 and 11 in the NRG1 isoform HRG-β2b. Panel C shows qPCR and relative changes in gene expression calculated using the 2−ΔΔCt method with β2-microglobulin (β2M) as a reference gene. Mean increase in transcripts in neurons was statistically significant (p<0.05). Error bar is SD based on 3 or 4 replicates, each done in triplicate.

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