Project description:Here we describe a method to target, multiplex and sequence full-length, native single-molecule the human mitochondrial genome utilizing the RNA-guided DNA endonuclease Cas9.
Project description:During acute myelosuppression or thrombocytopenia, bone marrow (BM) hematopoietic cells respond rapidly to replenish peripheral blood platelets. While the cytokine Thrombopoietin (Thpo) concomitantly regulates platelet production and maintains HSC stem cell potential whether Thpo directly controls Mk-lineage differentiation of HSCs is unclear. Stress hematopoiesis requires quiescent HSCs to proliferate, a process which depends on a higher energy production. However, whether this switch of metabolic state relates to lineage differentiation of HSCs is not known. We here show that Thpo rapidly upregulates mitochondrial activity in HSCs which was accompanied by preferential differentiation to Mk-lineage. During unperturbed hematopoiesis, HSCs with high mitochondrial content and activity exhibit Mk-lineage biased differentiation. Furthermore, Thpo rapidly skewed HSCs to express a tetraspanin molecule, CD9, which expression correlated to mitochondria cell content. While highly proliferative, mitochondrial-rich HSCs were resistant to apoptosis and oxidative stress upon Thpo stimulation. Thpo regulated mitochondrial activity did not associate with high levels of cMpl expression but was influenced by non-nuclear mitochondrial translocation of phosphorylated of STAT3 at serine 727. Our data reveals that HSCs are metabolically heterogeneous and higher mitochondrial activity primes HSCs toward the Mk lineage differentiation. We also uncover a pivotal role of Thpo in regulating the rapid Mk-lineage commitment during stress hematopoiesis. Our findings suggest that mitochondria metabolism primes HSCs not only to exit dormancy but toward direct differentiation to Mk lineage.
Project description:The human transcriptome is so large, diverse and dynamic that, even after a decade of investigation by RNA sequencing (RNA-Seq), we are yet to resolve its true dimensions. RNA-Seq suffers from an expression-dependent bias that impedes discovery of low-abundance transcripts and has prevented a complete census of gene expression. Here we performed targeted single-molecule and short-read RNA-Seq to survey the transcriptional landscape of a single human chromosome (Hsa21) at unprecedented resolution. Our analysis reaches the lower limits of the transcriptome and identifies a fundamental distinction between the architecture of protein-coding and noncoding gene content. Unlike their coding counterparts, noncoding exons undergo universal alternative splicing to produce a seemingly limitless variety of isoforms. Targeted RNA-Seq analysis of syntenic regions of the mouse genome shows that few noncoding exons are shared between human and mouse. Despite this divergence, human alternative splicing profiles are recapitulated on Hsa21 in mouse cells, indicative of regulation by a local splicing code that is more strongly conserved than the noncoding isoforms themselves. We propose that noncoding exons are functionally modular, with combinatorial alternative splicing generating an enormous repertoire of potential regulatory RNAs and a rich transcriptional reservoir for gene evolution.
