Variant U1 snRNAs are implicated in human pluripotent stem cell maintenance and neuromuscular disease

Abstract

The U1 small nuclear (sn)RNA (U1) is a multifunctional ncRNA, known for its pivotal role in pre-mRNA splicing and regulation of RNA 3' end processing events. We recently demonstrated that a new class of human U1-like snRNAs, the variant (v)U1 snRNAs (vU1s), also participate in pre-mRNA processing events. In this study, we show that several human vU1 genes are specifically upregulated in stem cells and participate in the regulation of cell fate decisions. Significantly, ectopic expression of vU1 genes in human skin fibroblasts leads to increases in levels of key pluripotent stem cell mRNA markers, including NANOG and SOX2. These results reveal an important role for vU1s in the control of key regulatory networks orchestrating the transitions between stem cell maintenance and differentiation. Moreover, vU1 expression varies inversely with U1 expression during differentiation and cell re-programming and this pattern of expression is specifically de-regulated in iPSC-derived motor neurons from Spinal Muscular Atrophy (SMA) type 1 patient's. Accordingly, we suggest that an imbalance in the vU1/U1 ratio, rather than an overall reduction in Uridyl-rich (U)-snRNAs, may contribute to the specific neuromuscular disease phenotype associated with SMA.

Figure 1.

vU1 gene expression is regulated throughout differentiation and cell re-programming. (A) Schematic of the protocol used for the gradual differentiation of human ESCs (HUES2) into monocytes. (B) Expression profiling of nascent vU1 levels in HUES2, Embryoid bodies (EB) and HUES2-derived monocytes, by quantitative reverse-transcription (qRT)-PCR analysis. vU1 levels were estimated using a gDNA as standard and normalized to 7SK levels across the different cell types. The position of the primers is indicated in the schematic. Regulatory elements, known to be required for U1/vU1 expression (proximal sequence element (PSE) and 3′ end processing (3′box)) are noted on the schematic. vU1 genes that show a greater than 95% sequence identity are grouped. Error bars represent standard error of the mean (SEM) of 3 independent differentiation experiments (two-way ANOVA analysis, * = P < 0.1, ** = P < 0.05, *** = P < 0.001, **** = P < 0.0001 and one-way ANOVA analysis (vU1.8 and vU1.3-5,12+20 genes only); * = P < 0.05. (C) Schematic of the protocol used for the de-differentiation of human skin fibroblasts into pluripotent stem cells. (D) Expression profiling of nascent vU1 levels in human skin fibroblasts (Fb) and fibroblast-derived induced pluripotent stem cells (iPSCs), by qRT-PCR analysis. Primers used are illustrated in the schematic as in (B). Error bars represent SEM of 3 independent re-programming experiments (two-way ANOVA analysis (vU1.7+9, vU1.13–16+19 and vU1.18); **** = P < 0.0001 and one-way ANOVA analysis; * = P < 0.05).

Figure 2.

vU1 and U1 genes follow reciprocal patterns of expression during differentiation and cell re-programming. (A) qRT-PCR analysis of steady state U1, vU1.8 and vU1.20 levels in human ESCs, EBs, ESC-derived monocytes, human skin fibroblasts and fibroblasts-derived iPSCs. Primers used are illustrated in the schematic above the graph. U1/vU1 levels were estimated using a genomic (g)DNA as standard and normalized to 7SK levels across the different cell types. U1 levels are indicated on the Y-axis to the left of the graph and vU1 levels on the right Y-axis. Error bars represent SEM of three independent differentiation/re-programming experiments (Two-way ANOVA analysis (U1); **** = P < 0.0001 and one-way ANOVA (vU1.8 +vU1.20); ** = P < 0.01, *P = 0.05. (B) The ratio of vU1 to U1 levels, expressed as a percentage of U1 levels, across the different cell types.

Figure 3.

vU1s participate in early cell fate decisions. (A) Steady state levels of pluripotent stem cell marker mRNAs, including OCT4, NANOG and SOX2, were measured following transfection of human fibroblasts (NHDF) cells with increasing doses (0.0375, 0.0625, 0.125 and 0.25 μg) of a mixed pool of vU1-expressing plasmids (vU1.2, vU1.3, vU1.8, vU1.13 and vU1.20), by qRT-PCR analysis. Changes in pluripotent mRNA levels (left graph), and vU1 levels (that could be specifically amplified) (right graph), are expressed as fold-difference over levels quantitated in cells transfected with control vector alone, which is set to 1.0. Error bars represent SEM of three independent transfection experiments (Two-way ANOVA analysis; ** = P > 0.05, *** = P > 0.001, **** = P > 0.0001). (B) FACS analysis of NANOG expression in human fibroblasts transfected with decreasing doses of the pooled vU1 plasmids, including 0.5, 0.25 and 0.125 μg. iPSCs and pGEM4 transfected human fibroblast (NDHF-1) cells were used as positive and negative controls, respectively. Histograms represent NANOG fluorescence (black line) compared to isotype control (shaded gray). The % of NANOG positive cells is noted in each histogram.

Figure 4.

Human vU1s are implicated in SMA. (A) qRT-PCR analysis of U1, vU1.8 and vU1.20 levels in healthy control (Ctl) and SMA patient's skin Fb, iPSCs and MNs. U1 levels are indicated on the Y-axis to the left of the graph and vU1 levels on the right Y-axis. Error bars represent SEM of 2 independent repeats (n = 4) (Two-way ANOVA analysis; **** = P > 0.0001 and one-way ANOVA analysis (vU1.8 and vU1.20 genes only); * = P < 0.05). (B) qRT-PCR analysis of U1, vU1.8 and vU1.20 levels in Ctl and Parkinson's patient skin Fb. iPSCs and DNs. U1 levels are indicated on the Y-axis to the left of the graph and vU1 levels on the right Y-axis. Error bars represent SEM of 2 independent repeats (n = 4) (Two-way ANOVA analysis; *** = P > 0.001, **** = P > 0.0001). (C) The levels of OCT4, TERT, NANOG, SMN (SMN-FL), CRABP1, FoxA2, TGF-β, CTNNBL1 and RRBP1 transcripts were measured in total RNAs extracted from SMA MNs and Parkinson's disease patient DNs. The magnitude of change (Fold difference (log2)) in SMA MNs and Parkinson disease DNs, relative to healthy controls MNs and DNs, respectively, as determined by qRT-PCR analysis. Levels were normalized to 18s rRNA across the different cell types. Error bars represent standard error of the mean (SEM) of 2 independent repeats (n = 4). (D) The magnitude of change of ATF6 and CASP3 transcripts (Fold difference (log2)) in SMA MNs and Parkinson disease DNs, relative to corresponding healthy controls MNs and DNs, respectively, as determined by qRT-PCR analysis. Levels were normalized to 18s rRNA across the different cell types. Error bars represent SEM of 2 independent repeats (n = 4).