Highlights d Modeling time-resolved scRNA-seq data avoids pitfalls of splicing RNA velocities d Dynamo reconstructs analytical vector fields from discrete velocity vectors d Vector fields reveal the timing and mechanisms of human hematopoiesis d Dynamo allows cell-state transition path and in silico perturbation predictions

Overexpressed in colon carcinoma-1 (OCC-1) is one of the earliest annotated long noncoding RNAs (lncRNAs) in colorectal cancer (CRC); however, its function remains largely unknown. Here, we revealed that OCC-1 plays a tumor suppressive role in CRC. OCC-1 knockdown by RNA interference promotes cell growth both in vitro and in vivo, which is largely due to its ability to inhibit G0 to G1 and G1 to S phase cell cycle transitions. In addition, overexpression of OCC-1 can suppress cell growth in OCC-1 knockdown cells. OCC-1 exerts its function by binding to and destabilizing HuR (ELAVL1), a cancer-associated RNA binding protein (RBP) which can bind to and stabilize thousands of mRNAs. OCC-1 enhances the binding of ubiquitin E3 ligase β-TrCP1 to HuR and renders HuR susceptible to ubiquitination and degradation, thereby reducing the levels of HuR and its target mRNAs, including the mRNAs directly associated with cancer cell growth. These findings reveal that lncRNA OCC-1 can regulate the levels of a large number of mRNAs at post-transcriptional level through modulating RBP HuR stability.

Related studies showed that the protein PSF represses protooncogene transcription, and VL30 -1 RNA, a mouse noncoding retroelement RNA, binds and releases PSF from a proto-oncogene, activating transcription. Here we show that this mechanism regulates tumorigenesis in human cells, with human RNAs replacing VL30 -1 RNA. A library of human RNA fragments was used to isolate, by affinity chromatography, 5 noncoding RNA fragments that bind to human PSF (hPSF), releasing hPSF from a protooncogene and activating transcription. T he protein PSF (1) contains a DNA-binding domain (DBD) that binds to the regulatory region of a proto-oncogene and represses transcription, and 2 RNA-binding domains (RBDs) that bind VL30-1 RNA, releasing PSF from a repressed protooncogene and activating transcription (2-5). Mouse and human genomes encode homologous PSF proteins with Ϸ95% sequence identity, whereas the VL30-1 gene belongs to a family of mouse noncoding retroelement genes (6) that is not present in the human genome (7). To determine whether the PSF/RNA regulatory mechanism functions in human cells, a library of RNA fragments was constructed from the nuclear RNA repertoire of a human tumor cell, and the library was screened by affinity chromatography for RNAs that bind to human PSF (hPSF). The screen identified 5 hPSF-binding noncoding RNA fragments that release hPSF from a repressed proto-oncogene and activate transcription, similar to VL30-1 RNA. Each human RNA fragment maps to a matching sequence in a different human gene. The following experiments show that human hPSF-binding RNAs are involved in the control of tumorigenesis. Results Cloning and Mapping Human RNA Fragments That Bind to hPSFProtein. The finding that VL30-1 RNA, a mouse retroelement RNA that is not encoded in the human genome, binds selectively to hPSF protein and reverses repression of proto-oncogene transcription (2-5), prompted a search for human RNAs that have a similar function as VL30-1 RNA. The procedure involved synthesizing a library of RNA fragments from the nuclear RNA repertoire of a human melanoma line and selecting by affinity chromatography RNA fragments that bind to hPSF. The procedure yielded 5 such RNA fragments, 4 of which were mapped, by sequence identity, within 1 of the following genes: L1PA16, a non-LTR retroelement gene (8); MER11C, a LTR retroelement gene (9); MALAT-1, a noncoding gene (10, 11); or HN, a mitochondrial gene coding for the peptide humanin (12); a fifth fragment, not shown in the figure, maps to a region that has not been characterized ( Fig. 1 and SI Text).The sequence of the HN RNA fragment is 100% identical to a sequence in the mitochondrial 16S ribosomal RNA gene and is 85% identical to positions 21947595-21947823 on nuclear chromosome 17. Further testing showed that the HN RNA fragment is derived from the mitochondrial HN RNA and not from the nuclear RNA (SI Text). The mitochondrial HN RNA might be translocated to the nucleus or derived from a mitochondrial contamination in the nuclear preparation.Release of hPSF from ...

The mammalian protein PSF contains a DNA-binding domain (DBD) that coordinately represses multiple oncogenic genes in human cell lines, indicating a role for PSF as a human tumor-suppressor protein. PSF also contains two RNA-binding domains (RBD) that form a complex with a noncoding VL30 retroelement RNA, releasing PSF from a gene and reversing repression. Thus, the DBD and RBD in PSF are linked by a mechanism of reversible gene regulation involving a noncoding RNA. This mechanism also could apply to other regulatory proteins that contain both DBD and RBD. The mouse genome has multiple copies of VL30 retroelements that are developmentally regulated, and mouse cells contain VL30 RNAs that have normal and pathological roles in gene regulation. Human chromosome 11 has a VL30 retroelement, and a VL30 EST was identified in human blastocyst cells, indicating that the PSF-VL30 RNA regulatory mechanism also could function in human cells.

We describe a mechanism of gene regulation involving formation of a complex between PSF protein and mouse VL30 (mVL30) retrotransposon RNA. PSF represses transcription of the insulin-like growth factor 1 (IGF1)-inducible gene P450scc by binding to an insulin-like growth factor response element (IGFRE) motif in the gene. The complex with mVL30 RNA releases PSF, allowing transcription to proceed. Retrovirally mediated transmission of mVL30 RNA to human tumor cells induced several genes, including oncogenes, which also are induced by IGF1, and promoted metastasis. In mice, steroid synthesis is activated in steroidogenic cells by pituitary hormones, which concomitantly induce transcription of mVL30 RNA in the cells. We showed that steroid synthesis could also be activated in mouse steroidogenic adrenal cells by transfection with cDNA encoding either mVL30 RNA tracts that form a complex with PSF or a small interfering RNA (siRNA) that degrades PSF transcripts. These results suggest that mVL30 RNA regulates steroidogenesis, and possibly other physiological processes of mice, by complex formation with PSF. Retrotransposons such as mVL30 apparently evolved not only as ''junk'' DNA but also as transcriptionally active noncoding DNA that acquired physiological and pathological functions. The complete genome of a mouse VL30 (mVL30) retrotransposon is structurally similar to a retroviral genome, with 5Ј and 3Ј LTRs flanking an internal region containing Ϸ3.7 kb (1). In contrast to the internal region of an infectious retroviral genome that encodes gag, pol, and env proteins, the corresponding region of the mVL30 genome has numerous stop codons in all reading frames that probably block formation of a functional protein. The mouse genome contains multiple copies of transcriptionally active mVL30 DNA, and virtually all mouse cells contain mVL30 RNA although the level varies among different tissues and at different developmental stages (1). Because retroviral vectors for gene transfer and gene therapy usually are produced in packaging cells derived from mouse cells, these cells also contain mVL30 RNA. A remarkable property of retroviral vectors is the capacity to transmit mVL30 RNA from a packaging cell to cells infected by the retrovirus, which synthesize, integrate, and transcribe mVL30 cDNA (1). In an earlier report (2), we showed that retroviral-mediated transfection of tissue factor (TF) cDNA into a nonmetastatic human melanoma cell line increased the metastatic potential of the cells to which mVL30-1 RNA also had been transmitted. The increase in metastatic potential depended on expression of TF protein on the cell surface and also on transcription of mVL30-1 cDNA. Dependence on mVL30-1 RNA was surprising because the RNA seems to lack significant coding potential. Here, we report that mVL30-1 RNA forms a complex with PSF, a multifunctional regulatory protein that binds to RNA in spliceosomes (3, 4) and to an IGFRE motif in the insulin-like growth factor 1 (IGF1)-inducible gene P450ssc that represses transcription of the g...

The concentrated autologous bone marrow containing mononuclear cells implantation relieves hip pain, prevents the progression of osteonecrosis. Therefore, it may be the treatment of choice particularly in stages I-II nontraumatic osteonecrosis of the femoral head.

cancer therapy ͉ Rab23 ͉ regulatory RNA ͉ tumor suppression P receding studies have described a reversible mechanism controlling gene transcription that involves PSF protein (1) and VL30-1 RNA, a member of the VL30 family of mouse retroelement noncoding RNAs (2, 3). PSF contains a DNAbinding domain (DBD) that binds and represses transcription of genes that have a PSF-binding site (4-6) and 2 RNA-binding domains (RBDs) that bind VL30-1 RNA, forming a PSF-RNA complex that dissociates from a gene and activates transcription (5-7). Increasing expression of PSF in a human tumor cell suppressed tumorigenesis (6), and ectopic expression of VL30-1 RNA in a human tumor cell promoted metastasis (3). VL30 RNAs are expressed in virtually all tissues of adult mice (8) and are associated with Ras-mediated transformation of mouse fibroblast cells (9). Here, we extend our studies of the function of PSF and VL30-1 RNA to the regulation of proto-oncogene transcription, cell proliferation, and tumorigenesis in mice. The results indicate that PSF is a major tumor-suppressor protein and VL30-1 RNA is a major tumor-promoter RNA in mice. ResultsExpression of PSF and VL30 -1 RNA in NIH/3T3 and B16F10 Cell Lines.NIH/3T3 and B16F10 cells were transfected with a transgene encoding PSF (NIH/3T3-PSF1 and B16F10-PSF1 lines) or VL30-1 RNA (NIH/3T3-VL301 line) or with a plasmid encoding a shRNA that causes degradation of PSF mRNA (NIH/ 3T3-PSF2 line) or VL30 -1 RNA (NIH/3T3-VL302 and B16F10-VL302 lines). The control lines were transfected with an empty plasmid pcDNA3.1(ϩ) (NIH/3T3-pcDNA3.1 and B16F10-pcDNA3.1 cell lines) or a plasmid encoding shLuciferase (NIH/3T3-shLuc and B16F10-shLuc lines). The NIH/ 3T3 and B16F10 cell lines were assayed for expression of PSF mRNA and VL30-1 RNA ( Fig. 1 and Table 1) and PSF protein (Fig. 1).Binding of PSF to the Regulatory DNAs of Mouse Genes. Chromatin fragments from NIH/3T3 cells were coimmunoprecipitated with anti-PSF antibody, and the DNAs in the fragments were tested for hybridization to a mouse gene-promoter chip (NimbleGen; Roche) that contains 385,000 regulatory DNAs from the mouse genome. A total of 57 DNA fragments were identified by chip assay, of which 12 mapped to characterized mouse coding genes [supporting information (SI) Tables S1 and S2]. The 12 DNAs bound to PSF in NIH/3T3 and B16F10 cells, as shown by a ChIP assay (Fig. 2); the P450scc gene had been identified as a PSF-binding gene in earlier studies (4, 6). We chose the Rab23 gene, a member the RAS gene family (10,11), to analyze the role of PSF and VL30-1 RNA in the regulation of proto-oncogene transcription in mice. (Table 1). Binding of Rab23 DNA to PSF is higher in NIH/3T3-PSF1 and B16F10-PSF1 cells and lower in NIH/3T3-PSF2 cells than in NIH/3T3 or B16F10 control cells (Fig. 3A). (ii) Transcription of Rab23 was analyzed in the same cell lines by RT-PCR. Transcription is lower in NIH/3T3-PSF1 and B16F10-PSF1 cells and higher in NIH/3T3-PSF2 cells than in NIH/3T3 or B16F10 control cells (Fig. 3B). The results indicate that PSF bin...

Background:The physiological function of cyclin K is poorly defined. Results: Cyclin K interacts with CDK12 and CDK13, and knockdown of cyclin K, CDK12, or CDK13 causes embryonic stem cell differentiation. Conclusion: Cyclin K, CDK12, and CDK13 are required for embryonic stem cell self-renewal. Significance: Novel kinase complexes are identified to maintain embryonic stem cell pluripotency.