Nearly all human multi-exonic genes undergo alternative splicing (AS) via regulation by RNA-binding proteins (RBPs), but few studies have examined the temporal dynamics of AS and its regulation during cell differentiation in the bone niche. We sought to evaluate how AS, under the control of RBPs, affects cell fate commitment during induced osteogenic differentiation of human bone marrow-derived multipotent stem/stromal progenitor cells (MSPCs). We generated a time-course RNA sequencing (RNA-seq) dataset representative of induced MSPC differentiation to osteoblasts. Our analysis revealed widespread AS changes, coordinated with differential RBP expression, at multiple time points, including many AS changes in non-differentially expressed genes. We also developed a computational approach to profile the dynamics and regulation of AS by RBPs using time-course RNA-seq data, by combining temporal patterns of exon skipping and RBP expression with RBP binding sites in the vicinity of regulated exons. In total we identified nine RBPs as potential key splicing regulators during MSPC osteogenic differentiation. Perturbation of one candidate, KHDRBS3, inhibited osteogenesis and bone formation in vitro, validating our computational prediction of "driver" RBPs. Overall, our work highlights a high degree of complexity in the splicing regulation of MSPC osteogenic differentiation. Our computational approach may be applied to other time-course data to explore dynamic AS changes and associated regulatory mechanisms in other biological processes or disease trajectories.

Picea mongolica is a rare tree species in China, which is of great significance in combating desertification and improving the harsh ecological environment. Due to the low rate of natural regeneration, high mortality, and susceptibility to pests and cold springs, Picea mongolica has gradually become extinct. At present, somatic embryogenesis (SE) is the most effective method of micro-proliferation in conifers, but the induction rate of embryogenic callus (EC) is low, and EC is difficult to differentiate from non-embryonic callus (NEC). Therefore, the EC and NEC of Picea mongolica were compared from the morphology, histological, physiological, and transcriptional levels, respectively. Morphological observation showed that the EC was white and transparent filamentous, while the NEC was compact and brownish-brown lumpy. Histological analyses showed that the NEC cells were large and loosely arranged; the nuclei attached to the edge of the cells were small; the cytoplasm was low; and the cell gap was large and irregular. In the EC, small cells, closely arranged cells, and a large nucleus and nucleolus were observed. Physiological studies showed significant differences in ROS-scavenging enzymes between the EC and NEC. Transcriptome profiling revealed that 13,267 differentially expressed genes (DEGs) were identified, 3682 were up-regulated, and 9585 were down-regulated. In total, 63 GO terms had significant enrichment, 32 DEGs in plant hormone signal transduction pathway were identified, and 502 different transcription factors (TFs) were characterized into 38 TF families. Meanwhile, we identified significant gene expression trends associated with somatic embryo development in plant hormones (AUX/IAA, YUCCA, LEA, etc.), stress (GST, HSP, GLP, etc.), phenylpropanoid metabolism (4CL, HCT, PAL, etc.), and transcription factors (AP2/ERF, MYB, WOX, etc.). In addition, nine genes were chosen for RT-qPCR, and the results were consistent with RNA-Seq data. This study revealed the changes in morphology, histology, physiology, and gene expression in the differentiation of NEC into EC and laid the foundation for finding the key genes to promote EC formation.