Barbara Cisterna

Small ribosomal subunits associate with nuclear myosin and actin in transit to the nuclear pores

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 ∼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.—Cisterna, B., Necchi, D., Prosperi, E., Biggiogera, M. Small ribosomal subunits associate with nuclear myosin and actin in transit to the nuclear pores. FASEB J. 20, E1257–E1263 (2006)

Apoptosis-linked changes in the phosphorylation status and subcellular localization of the spliceosomal autoantigen U1-70K.

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.

Ribosome biogenesis: from structure to dynamics.

In this chapter we describe the status of the research concerning the nucleolus, the major nuclear body. The nucleolus has been recognized as a dynamic organelle with many more functions than one could imagine. In fact, in addition to its fundamental role in the biogenesis of preribosomes, the nucleolus takes part in many other cellular processes and functions, such as the cell-cycle control and the p53 pathway: the direct or indirect involvement of the nucleolus in these various processes makes it sensitive to their alteration. Moreover, it is worth noting that the different nucleolar factors participating to independent mechanisms show different dynamics of association/disassociation with the nucleolar body.

Perichromatin fibrils accumulation in hepatocyte nuclei reveals alterations of pre-mRNA processing during aging

We previously described an unusual accumulation of perichromatin fibrils (PF)-the in situ form of pre-mRNA transcription and early splicing-in hepatocyte nuclei of old rats. Here we have investigated, by immunoelectron microscopy, the nature of such PF, analyzing the presence of transcription, splicing and cleavage factors, polyadenylated RNA, and the incorporation of bromouridine in adult and old rats. Our observations revealed alterations in amount and/or distribution of pre-mRNA transcription, splicing and cleavage factors, as well as of polyadenylated RNA, together with lower bromouridine incorporation in newly transcribed RNA in the hepatocyte nucleoplasm of old rats. Therefore, our data indicate both a decrease in pre-mRNA transcription and a slow down of PF processing and transport during aging.

An active mechanism flanks and modulates the export of the small ribosomal subunits

The modalities of export of the ribosomal subunits from the nucleolus to the nuclear pores have been only partially clarified since it is not yet clear whether the movements depend purely on diffusion or also from an active process. Recently, we suggested the existence of an active transport mechanism of a subset (10–12%) of the small ribosomal subunits (SSU) (Cisterna et al. in 2006, Faseb J). Here, we give further evidence that an active, motor protein-mediated process exists for the SSU transport from the nucleolus to the nuclear pore. We demonstrate that the blockade of ATP synthesis and antibody-mediated inhibition of nuclear myosin or actin induce structural and functional modifications of the nucleolus, suggestive of transcriptional activity decrease. Moreover, both treatments induce a significant retention of RNA inside the nucleus and an accumulation of ribosomal subunits in the granular component. We suggest that the existence of this secondary, active mechanism of SSU transport might be utilized by the cell when a more rapid and directional export is needed.

Can a genetically-modified organism-containing diet influence embryo development? A preliminary study on pre-implantation mouse embryos

In eukaryotic cells, pre-mRNAs undergo several transformation steps to generate mature mRNAs. Recent studies have demonstrated that a diet containing a genetically modified (GM) soybean can induce modifications of nuclear constituents involved in RNA processing in some tissues of young, adult and old mice. On this basis, we have investigated the ultrastructural and immunocytochemical features of pre-implantation embryos from mice fed either GM or non- GM soybean in order to verify whether the parental diet can affect the morpho-functional development of the embryonic ribonucleoprotein structural constituents involved in pre-mRNA pathways. Morphological observations revealed that the general aspect of embryo nuclear components is similar in the two experimental groups. However, immunocytochemical and in situ hybridization results suggest a temporary decrease of pre-mRNA transcription and splicing in 2-cell embryos and a resumption in 4-8-cell embryos from mice fed GM soybean; moreover, pre-mRNA maturation seems to be less efficient in both 2-cell and 4-8-cell embryos from GM-fed mice than in controls. Although our results are still preliminary and limited to the pre-implantation phases, the results of this study encourage deepening on the effects of food components and/or contaminants on embryo development.

Discrete foci containing RNAse A are found in nucleoli of HeLa cells after aging in culture

We have studied by means of ultrastructural immunocytochemistry the localization of RNase A in nuclei of HeLa cells in control conditions and following cell ageing in culture. We have found that roundish, electron dense foci, which contain a significant amount of RNase A, can be detected within nucleoli of aged cells. These bodies also contain RNA and lack ribosomal S3 proteins, and may represent either simple storage sites or areas where RNA degradation takes place.

Use of halogenated precursors to define a transcription time window after treatment with hypometabolizing molecules

Abstract A new method is proposed which combines the high spatial resolution of transmission electron microscopy with information on the dynamics of transcription. Incorporation of two different RNA precursors was used to define a time transcription window on cultured cells treated with hypometabolizing peptides which are known to modulate transcription. This procedure allows detecting a single fibril of newly synthesized RNA in the time range in which it is transcribed.

Effect of adapted physical exercise on satellite cells from skeletal muscles of old mice: ex vivo and in vitro analyses

Aging is characterized by loss of skeletal muscle mass termed sarcopenia, which contributes to frailty, disability and premature death (1). The mechanisms leading to sarcopenia are manifold, the decline in muscle regeneration efficiency playing a crucial role. In the absence of specific therapies, studies have stressed the importance of physical exercise as an effective approach to prevent/limit the sarcopenic drive (2,3). We investigated the effect of adapted exercise on the number and myogenic properties of satellite cells (SCs) in the quadriceps femoris and gastrocnemius muscles of old mice (28 months). Both muscles contain about 90% of type II fibers, which are especially affected by sarcopenia (4). We compared old exercised mice with old sedentary mice, adult sedentary mice (12 months) being the control. SCs were identified and quantified ex vivo; the proliferation and differentiation potential of SC-derived myoblasts from the three groups of mice was studied in vitro. Ultrastructural morphology and immunocytochemistry at light and electron microscopy level localized molecular markers of SC activation and protein factors involved in RNA transcription and splicing. Results showed that: 1) physical exercise induces an increase in the total number as well as activated fraction of SCs compared with sedentary old specimens; 2) myoblasts from exercised old muscles show morphological features and nuclear activity quite similar to myoblasts from adult subjects, whereas myoblasts from non-exercised old muscles exhibit structural and functional alterations suggestive of a reduced metabolic activity; 3) myotubes differentiated from myoblasts of exercised old muscles resemble those from adult myoblasts, whereas myotubes from non-exercised old muscles show marked structural alterations. Physical exercise induces numerical increase and activation of SCs in old muscles and improves their capability to differentiate into structurally and functionally complete myotubes. Adapted physical exercise may represent a non-pharmacological approach to stimulate SCs, enhancing muscle quality at very advanced age. MC is a PhD student of the Doctoral Program “Multimodal Imaging in Biomedicine” (University of Verona).

Magnetic resonance imaging investigations in a murine model of Down syndrome: the Ts65Dn mouse

Trisomy 21, or Down’s syndrome, is the most common genetic cause of intellectual disability; moreover, DS patients suffer from muscle hypotonia and low muscle strength whose mechanisms are still unknown and only partially explained by intellectual disability. Genetic mouse models (e.g., the Ts65Dn mouse) may offer insights into the responsible mechanisms . Moreover, the Ts65Dn mouse show muscle weakness and share morphological similarities with sarcopenia of aging [1]. To characterize skeletal muscle in theTs65Dn model, we explored the hindlimb of young adult (6-month-old) mice in euploid (n=9, mean weight: 43.8±4.10 g) and trisomic (n=9, mean weight: 38.2±4.0 g ) individuals by magnetic resonance imaging. Mice were imaged in a Bruker Tomograph at 4.7 Tesla using a RARE T2-weighed sequence (TR: 5000ms, TE: 56ms; spatial resolution 0.0182cm2 per pixel). Muscle cross sectional area was measured in a section taken at mid-femur in either hindlimbs and values averaged for further analysis. Results showed that mean hindlimbs muscle cross sectional area was larger in trisomic than in euploid mice (0.29±0.05 vs. 0.23±0.05 cm2, p=0.03 [t test]). Because of significant difference in body weight between euploid and trisomic mice (p=0.009), data were adjusted per body weight. Using adjusted data, a significant difference was still found between groups (euploid: 0.005±0.001; trisomic: 0.007±0.001 cm2/g body weight). It is concluded that young adult trisomic mice has increased apparent muscle mass in hindlimbs. Since trisomic Ts65Dn mice show reduced grip strength as well as running and swimming speed in the presence of roughly normal muscle biochemistry [2], possible structural and fibertype changes in hindlimb muscles of these mice are under investigation to explain such a discrepancy.