Microvesicles (MVs) play a pivotal role in cell-to-cell communication. Recent studies demonstrated that MVs may transfer genetic information between cells. Here, we show that MVs derived from human adult liver stem cells (HLSC) may reprogram in vitro HepG2 hepatoma and primary hepatocellular carcinoma cells by inhibiting their growth and survival. In vivo intratumor administration of MVs induced regression of ectopic tumors developed in SCID mice. We suggest that the mechanism of action is related to the delivery of microRNAs (miRNAs) from HLSC-derived MVs (MV-HLSC) to tumor cells on the basis of the following evidence: (a) the rapid, CD29-mediated internalization of MV-HLSC in HepG2 and the inhibition of tumor cell growth after MV uptake; (b) the transfer by MV-HLSC of miRNAs with potential antitumor activity that was downregulated in HepG2 cells with respect to normal hepatocytes; (c) the abrogation of the MV-HLSC antitumor effect after MV pretreatment with RNase or generation of MVs depleted of miRNAs; (d) the relevance of selected miRNAs was proven by transfecting HepG2 with miRNA mimics. The antitumor effect of MV-HLSC was also observed in tumors other than liver such as lymphoblastoma and glioblastoma. These results suggest that the delivery of selected miRNAs by MVs derived from stem cells may inhibit tumor growth and stimulate apoptosis. Stem Cells2012;30:1985–1998

Mammalian cleavage factor I (CF I m ) is an essential factor that is required for the first step in pre-mRNA 3 end processing. Here, we characterize CF I m 68 subnuclear distribution and mobility. Fluorescence microscopy reveals that in addition to paraspeckles CF I m 68 accumulates in structures that partially overlap with nuclear speckles. Analysis of synchronized cells shows that CF I m 68 distribution in speckles and paraspeckles varies during the cell cycle. At an ultrastructural level, CF I m 68 is associated with perichromatin fibrils, the sites of active transcription, and concentrates in interchromatin granules-associated zones. We show that CFI m 68 colocalizes with bromouridine, RNA polymerase II, and the splicing factor SC35. On inhibition of transcription, endogenous CF I m 68 no longer associates with perichromatin fibrils, but it can still be detected in interchromatin granules-associated zones. These observations support the idea that not only splicing but also 3 end processing occurs cotranscriptionally. Finally, fluorescence recovery after photobleaching analysis reveals that the CF I m 68 fraction associated with paraspeckles moves at a rate similar to the more dispersed molecules in the nucleoplasm, demonstrating the dynamic nature of this compartment. These findings suggest that paraspeckles are a functional compartment involved in RNA metabolism in the cell nucleus. INTRODUCTIONMost functional mRNAs of eukaryotic genes are generated from their primary transcripts (pre-mRNAs) through RNA splicing and 3Ј end polyadenylation. Removal of introns occurs in the spliceosome, a complex composed of five small ribonucleoprotein particles (U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles [snRNPs]) and many non-snRNPs splicing factors, including members of the arginine-serine (SR) family of proteins. The mature 3Ј ends of mRNAs are generated by endonucleolytic cleavage of the pre-mRNA followed by polyadenylation of the upstream cleavage product. Biochemical studies have identified six factors required for efficient processing in vitro: the cleavage and polyadenylation specificity factor (CPSF), the cleavage stimulation factor (CstF), and two cleavage factors, mammalian cleavage factor I m [CF I m ] and CF II m , are necessary for the cleavage reaction. Polyadenylation requires in addition to CPSF, poly(A) polymerase, and the nuclear poly(A) binding protein 1 (PABPN1, previously called PAB II, for review, see Wahle and Rü egsegger, 1999). Other proteins involved in either transcription, such as the carboxy-terminal domain of RNA polymerase II, or capping (nuclear cap-binding complex) and splicing (U2AF65) have been shown to greatly enhance the efficiency of the first step of the reaction (Flaherty et al., 1997;Hirose and Manley, 1998;Millevoi et al., 2002).The mammalian cell nucleus is a highly structured and dynamic compartment. Many nuclear factors are localized in morphologically well-defined structural units that include the nucleolus and several "nuclear bodies," such as the Cajal ...

Mutations of MYH9, the gene for non-muscle myosin heavy chain IIA (NMMHC-IIA), cause a complex clinical phenotype characterized by macrothrombocytopenia and granulocyte inclusion bodies, often associated with deafness, cataracts and/or glomerulonephritis. The pathogenetic mechanisms of these defects are either completely unknown or controversial. In particular, it is a matter of debate whether haploinsufficiency or a dominant-negative effect of mutant allele is responsible for hematological abnormalities. We investigated 11 patients from six pedigrees with different MYH9 mutations. We evaluated NMMHC-IIA levels in platelets and granulocytes isolated from peripheral blood and in megakaryocytes (Mks) cultured from circulating progenitors. NMMHC-IIA distribution in Mks and granulocytes was also assessed. We demonstrated that all the investigated patients had a 50% reduction of NMMHC-IIA expression in platelets and that a similar defect was present also in Mks. In subjects with R1933X and E1945X mutations, the whole NMMHC-IIA of platelets and Mks was wild-type. No NMMHC-IIA inclusions were observed at any time of Mk maturation. In granulocytes, the extent of NMMHC-IIA reduction in patients with respect to control cells was significantly greater than that measured in platelets and Mks, and we found that wild-type protein was sequestered within most of the NMMHC-IIA inclusions. Altogether these results indicate that haploinsufficiency of NMMHC-IIA in megakaryocytic lineage is the mechanism of macrothrombocytopenia consequent to MYH9 mutations, whereas in granulocytes a dominant-negative effect of mutant allele is involved in the formation of inclusion bodies. The finding that the same mutations act through different mechanisms in different cells is surprising and requires further investigation.

We have followed at high resolution the ribosomal protein S6 entering the nucleus of HeLa cells, stopping in some (not all) interchromatin granules clusters and reaching, via Cajal bodies, the nucleolus. There, S6 is assembled with other proteins and rRNA into small ribosomal subunit (SSU), released in the nucleoplasm, and exported through the nuclear pores. We show for the first time the spatial association of nuclear myosin I (NMI) and actin with the SSU already at the nucleolar periphery to the nuclear pore. A blockade of NMI or actin induces an upstream accumulation of the S6 protein en route to the nucleolus, and a temperature lower than normal influences RNA export. Our data strongly suggest a functional relationship of SSU with NMI and actin. In our hypothesis, an active, myosin-driven movement of the small ribosomal subunit can be responsible for the export of approximately 10% of SSUs. This hypothesis is supported by ultrastructural, immunofluorescence, and biochemical analyses. The currently accepted model for the subunit release suggests a diffusive, temperature-independent mechanism. However, the advantage of the double mechanism would assure that the movement of a part of the subunits could be modulated, increased, or decreased according to the needs of the cell at a specific moment in the cell cycle.

Apoptosis consists of highly regulated pathways involving post-translational modifications and cleavage of proteins leading to sequential inactivation of the main cellular processes. Here, we focused on the apoptotic processing of one of the essential components of the mRNA splicing machinery, the U1-70K snRNP protein. We found that at an early stage of apoptosis, before the cleavage of the C-terminal part of the protein by caspase-3, the basal phosphorylation of the Ser140 residue located within the RNA recognition motif, increases very significantly. A caspase-dependent, PP1-mediated dephosphorylation of other serine residues takes place in a subset of U1-70K proteins. The U1-70K protein phosphorylated at Ser140 is clustered in heterogeneous ectopic RNP-derived structures, which are finally extruded in apoptotic bodies. The elaborate processing of the spliceosomal U1-70K protein we identified might play an important role in the regulated breakdown of the mRNA splicing machinery during early apoptosis. In addition, these specific changes in the phosphorylation/dephosphorylation balance and the subcellular localization of the U1-70K protein might explain why the region encompassing the Ser140 residue becomes a central autoantigen during the autoimmune disease systemic lupus erythematosus. Cell Death and Differentiation (2008) In higher eukaryotes, the process of gene expression includes transcription, pre-mRNA splicing/processing and translation in the cytoplasm. The macromolecular machinery involved in pre-mRNA splicing, the spliceosome, consists of five small nuclear ribonucleoparticles (snRNPs) called U1, U2 and U4-6 snRNP, and a large number of additional splicing factors. 1 Splicing factors are supposedly recruited from storage sites, that is interchromatin granules, in the interchromatin space to transcription sites at the periphery of condensed chromatin. The assembly of the spliceosome is initiated by recognition of the 5 0 splice site of pre-mRNA by U1 snRNP and mediated by serine/arginine-rich (SR) proteins. 2 Phosphorylation and dephosphorylation both have an important role in the recruitment of splicing factors and the subsequent regulation of spliceosome assembly and splicing catalysis. 3 The U1-snRNP-specific U1-70K protein is a highly phosphorylated protein, which contains two SR domains. Phosphorylation of both the SR protein ASF/SF2 and the first SR domain of the U1-70K protein are involved in vitro in their interaction. 4,5 In addition, thiophosphorylation of the U1-70K protein, making it resistant to dephosphorylation by phosphatases, results in complete inhibition of splicing, but not of spliceosome formation. 6 At least 11-13 natural variants of the U1-70K protein, partially due to phosphorylation, have been found by 2D analysis. 7,8 However, very little is known about specific phosphorylated residues in this protein or specific enzymes involved in the functional regulation of the U1-70K protein by phosphorylation/dephosphorylation.Apoptosis is a fast and orderly process consisting of a seq...

Chitosan-based nanoparticles (chiNPs) are considered to be potentially good carriers for the sustained intracellular delivery of specific molecules. However, scarce attention has been paid to the long-lasting permanence of these NPs in the intracellular milieu, as well as to their intracellular fate (i.e., distribution, interaction with cell organelles, and degradation) in the long term. In the present study, the presence and subcellular location of FITC-labelled chiNPs were monitored in HeLa cells up to 14 days post-administration using multicolorfluorescence confocal microscopy and diaminobenzidine photo-oxidation at transmission electron microscopy. The main result of the present study is the demonstration that internalized chiNPs persist inside the cell up to two weeks, occurring in both the cytoplasm and nucleus; accordingly, chiNPs are able to pass from mother to daughter cells through several mitotic cycles. The cells did not show increased mortality or structural damage up to 14 days after chiNP exposure.

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During ageing, a progressive loss of skeletal muscle mass and a decrease in muscle strength and endurance take place, in the condition termed sarcopenia. The mechanisms of sarcopenia are complex and still unclear; however, it is known that muscle atrophy is associated with a decline in the number and/or efficiency of satellite cells, the main contributors to muscle regeneration. Physical exercise proved beneficial in sarcopenia; however, knowledge of the effect of adapted physical exercise on the myogenic properties of satellite cells in aged muscles is limited. In this study the amount and activation state of satellite cells as well as their proliferation and differentiation potential were assessed in situ by morphology, morphometry and immunocytochemistry at light and transmission electron microscopy on 28-month-old mice submitted to adapted aerobic physical exercise on a treadmill. Sedentary age-matched mice served as controls, and sedentary adult mice were used as a reference for an unperturbed control at an age when the capability of muscle regeneration is still high. The effect of physical exercise in aged muscles was further analysed by comparing the myogenic potential of satellite cells isolated from old running and old sedentary mice using an in vitro system that allows observation of the differentiation process under controlled experimental conditions. The results of this ex vivo and in vitro study demonstrated that adapted physical exercise increases the number and activation of satellite cells as well as their capability to differentiate into structurally and functionally correct myotubes (even though the age-related impairment in myotube formation is not fully reversed): this evidence further supports adapted physical exercise as a powerful, non-pharmacological approach to counteract sarcopenia and the age-related deterioration of satellite cell capabilities even at very advanced age.