Journal: Journal of Molecular Cell Biology

Article Title: Single-cell landscape of alternative polyadenylation in human lymphoid hematopoiesis

doi: 10.1093/jmcb/mjae027

Figure Lengend Snippet: Global APA analysis reveals a trend toward 3′UTR shortening during lymphoid differentiation. ( A ) Schematic illustration of the pPUI calculation. C proximal is the read count of the proximal peak, and 〈C + 1〉 is the geometric mean of the counts of all the peaks associated with the 3′UTR. ( B ) Average pPUI of the 3188 multiple-PAS genes in each lymphoid cell population. The t -test was performed to determine the difference in pPUI between adjacent populations in the differentiation trajectory. * P < 0.05; ** P < 0.01; *** P < 0.001. ( C ) Cumulative distribution curve of the pPUI of 2364 dynamic-APA genes in 12 lymphoid cell populations. The square on the upper left shows partial magnification. The statistical significance between adjacent clusters during lymphoid differentiation was as follows: HSC vs. CMP.LMPP, P = 0.065; CMP.LMPP vs. CLP, P = 0.098; CLP vs. Pre.B, P = 0.0008; CLP vs. CD4.N, P = 0.591; CLP vs. CD8.N, P = 0.207; CLP vs. NK, P = 0.012; CLP vs. pDC, P = 0.022; Pre.B vs. B, P = 0.128; CD4.N vs. CD4.M, P = 0.027; CD8.N vs. CD8.CM, P = 0.091; CD8.N vs. CD8.EM, P = 0.003. ( D ) A schematic diagram illustrating the number of 3′UTRs containing two PASs that undergo shortening or lengthening between adjacent populations during the process of lymphoid differentiation. ( E ) The average pPUI of multiple-PAS genes (left) or dynamic-APA genes (right) was calculated at the single-cell level and projected onto the transcriptome clustering map. ( F ) The density distribution of the poly(A) signal AAUAAA in proximity to frequent (with the highest expression) and infrequent (with the lowest expression) PASs of 3′UTR exhibiting dynamic-APA events in HSCs and CD8.EM cells, respectively. The Kolmogorov–Smirnov test was used to demonstrate the distribution difference between frequent PASs and infrequent PASs in the region located 100 nt upstream and 100 nt downstream from the 3′ edge of the identified PAS peak.

Article Snippet: Human CD8.N, CD8.CM, and CD8.EM cells were isolated using magnetic beads with the EasySep Human Naïve CD8 + T Cell and Memory CD8 + T-Cell Enrichment Kits, respectively (STEMCELL Technologies), according to the manufacturer's instructions.

Techniques: Expressing

Journal: Journal of Molecular Cell Biology

Article Title: Single-cell landscape of alternative polyadenylation in human lymphoid hematopoiesis

doi: 10.1093/jmcb/mjae027

Figure Lengend Snippet: Specific 3′UTR shortening/lengthening in each cell population during lymphoid differentiation. ( A ) pPUI heatmap of genes with specific 3′UTR shortening/lengthening in the 12 lymphoid cell populations. Higher pPUI indicates specifically shortened 3′UTR, while lower pPUI indicates specifically lengthened 3′UTR. For populations in which the number of cell type-specific APA events was insufficient for GO enrichment, genes associated with lymphoid hematopoiesis and immune function in health and disease are labeled on the right. ( B ) Schematic illustration of the ratio of specific 3′UTR shortening/lengthening in each population. ( C ) Case visualization of cell type-specific APA events. Left, UBIAD1 exhibits 3′UTR lengthening in HSCs and shortening in terminally differentiated cells; middle, SDE2 exhibits 3′UTR shortening in CD8.EM cells; right, COPZ1 exhibits 3′UTR shortening in pDCs. P, proximal end of the 3′UTR; D, distal end of the 3′UTR. ( D ) GO enrichment analysis of cell type-specific 3′UTR-shortening APA events in CD8.EM cells, pDCs, and Pre.B cells. ( E ) GO enrichment analysis of cell type-specific 3′UTR-lengthening APA events in HSCs, CMP.LMPP cells, CD8.EM cells, and pDCs.

Article Snippet: Human CD8.N, CD8.CM, and CD8.EM cells were isolated using magnetic beads with the EasySep Human Naïve CD8 + T Cell and Memory CD8 + T-Cell Enrichment Kits, respectively (STEMCELL Technologies), according to the manufacturer's instructions.

Techniques: Labeling

Journal: Journal of Molecular Cell Biology

Article Title: Single-cell landscape of alternative polyadenylation in human lymphoid hematopoiesis

doi: 10.1093/jmcb/mjae027

Figure Lengend Snippet: 3′UTR APA patterns reflect lymphoid differentiation stages. ( A ) Single-cell clustering using the pPUI expression matrix-generated 14 populations. ( B ) pPUI single-cell clustering labeled with cell clusters based on RNA expression profiles. ( C ) The composition of RNA-based cell clusters within each pPUI-based cluster. ( D ) Pearson's correlation and clustering analyses of the pPUI expression matrix of the 12 populations. ( E ) Pearson's correlation and clustering analyses of RNA expression profiles of the 12 populations. ( F ) Pseudotime analysis showing the differentiation trajectory based on RNA expression profiles. ( G ) Heatmap displaying stage-specific APA events based on pPUI values. Higher pPUI indicates specifically shortened 3′UTR, while lower pPUI indicates specifically lengthened 3′UTR. ( H ) Visual representation of stage-specific APA genes. Left: IFITM3 , a gene with a lengthened 3′UTR in the HSPC stage; right: GTF2F2 , a gene with a shortened 3′UTR in the HSPC stage. ( I ) Differential expression analysis of eight APA core regulators between HSPCs (HSCs, CMP.LMPP cells, and CLP cells) and mature lymphocytes (NK, CD8.EM, B, CD8.N, CD4.N, CD4.M, and CD8.CM cells). Vertical dashed line, P = 0.05; horizontal dashed line, average fold change = 1.

Article Snippet: Human CD8.N, CD8.CM, and CD8.EM cells were isolated using magnetic beads with the EasySep Human Naïve CD8 + T Cell and Memory CD8 + T-Cell Enrichment Kits, respectively (STEMCELL Technologies), according to the manufacturer's instructions.

Techniques: Expressing, Generated, Labeling, RNA Expression, Quantitative Proteomics

Journal: Journal of Molecular Cell Biology

Article Title: Single-cell landscape of alternative polyadenylation in human lymphoid hematopoiesis

doi: 10.1093/jmcb/mjae027

Figure Lengend Snippet: Differential 3′UTR APA analysis during the peripheral differentiation of T cells. ( A ) Cumulative distribution curve of the pPUI values of dynamic-APA genes during peripheral T cell differentiation. The Kruskal–Wallis test was used to determine the statistical significance of differences. Left, CD4.N vs. CD4.M; middle, CD8.N vs. CD8.CM; right, CD8.N vs. CD8.EM. ( B ) Venn plot showing the differential APA events between naïve and memory T cells. The gene set of interest is outlined in red. ( C ) GO enrichment analysis of the gene set of interest. ( D – F ) Case visualization of differential APA events related to immunological function ( D ), lymphocyte function ( E ), and protein ubiquitination ( F ) during peripheral differentiation. q-val, adjusted P -value. Purple dashed box, PAS with significant change; blue coloring, clusters with significant PAS usage in the naïve T cell stage; pink coloring, clusters with significant PAS usage in the memory T cell stage; green arrow indicates the trend of APA change in the CD4 + T cell lineage; brown arrow indicates the trend of APA change in the CD8 + T cell lineage.

Article Snippet: Human CD8.N, CD8.CM, and CD8.EM cells were isolated using magnetic beads with the EasySep Human Naïve CD8 + T Cell and Memory CD8 + T-Cell Enrichment Kits, respectively (STEMCELL Technologies), according to the manufacturer's instructions.

Techniques: Cell Differentiation, Ubiquitin Proteomics

Journal: Journal of Molecular Cell Biology

Article Title: Single-cell landscape of alternative polyadenylation in human lymphoid hematopoiesis

doi: 10.1093/jmcb/mjae027

Figure Lengend Snippet: In vitro validation of the functional 3′UTR PAS switch in peripheral CD8 + lymphocytes. ( A ) Schematic illustration of the comparison of RE pPAS between different clusters. p1, proximal PAS in cell 1; p2, proximal PAS in cell 2; d1, distal PAS in cell 1; d2, distal PAS in cell 2. ( B ) RE pPAS of the immune-related genes CD5, GOLT1B , and TMEM59 . ( C ) RE pPAS of the lymphoid functional genes NFATC2IP, BCL2, TCF7 , and CD28 . ( D ) RE pPAS of the protein ubiquitination-related genes SUMO3, UBE2G1 , and YPEL5 . * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human CD8.N, CD8.CM, and CD8.EM cells were isolated using magnetic beads with the EasySep Human Naïve CD8 + T Cell and Memory CD8 + T-Cell Enrichment Kits, respectively (STEMCELL Technologies), according to the manufacturer's instructions.

Techniques: In Vitro, Biomarker Discovery, Functional Assay, Comparison, Ubiquitin Proteomics