Expression of transduced Nucleolin promotes the clearance of accumulated alpha-synuclein

Background Alpha-synuclein (αSyn) is a major component of Lewy bodies, which are known to be a pathogenic marker of Parkinson’s disease (PD). The accumulation of αSyn is caused by dysfunctions in protein degradation machinery. The reinforcement of αSyn degradation is a potential therapeutic target of PD since accumulated αSyn is responsible for the pathogenesis of PD. Nucleolin (NCL) is essential for forming nucleolar structure. The function of NCL is correlated with oxidative stress-mediated cell death. A previous study demonstrated that NCL was reduced in PD brains, and overexpression of NCL alleviated rotenone-induced neural toxic effects. Knockdown of NCL had the opposite effect. These results suggest that the malfunctioning of NCL would exacerbate PD pathology. Thus, we hypothesized that the introduction of ectopic NCL could rescue the α-synucleinopathy in PD. Methods We tested whether ectopic expression of NCL facilitates the clearance of αSyn. The Ectopic expression of NCL was accomplished by the transfection of GFP or GFP-NCL in mouse embryonic fibroblasts (MEF) or transduction of green fluorescent protein (GFP) or GFP-NCL using lentivirus in rat primary cortical neuron. We also investigated whether the expression of GFP or GFP-NCL in mouse substantia nigra alleviates the PD pathology derived by αSyn aggregates. Results The expression of NCL enhanced the clearance of αSyn accumulation or aggregates in MEF and rat primary cortical neurons. The activity of autophagy-lysosome pathway was enhanced by NCL expression. The transduction of NCL in the substantia nigra, which was co-injected with αSyn fibrils, rescued PD manifestations. Conclusions The elevation of NCL levels may reflect a therapeutic strategy for α-synucleinopathy in PD.

Inhibitor of Growth 4 (ING4) is a positive regulator of rRNA synthesis

Ribosome biogenesis is essential for maintaining basic cellular activities although its mechanism is not fully understood. Inhibitor of growth 4 (ING4) is a member of ING family while its cellular functions remain controversial. Here, we identified several nucleolar proteins as novel ING4 interacting proteins. ING4 localized in the nucleus with strong accumulation in the nucleolus through its plant homeodomain, which is known to interact with histone trimethylated H3K4, commonly present in the promoter of active genes. ING4 deficient cells exhibited slower proliferation and the alteration in nucleolar structure with reduced rRNA transcription, which was rescued by exogenous expression of GFP-ING4 to the similar levels of wild type cells. In the ING4 deficient cells, histone H3K9 acetylation and the key rRNA transcription factor UBF at the promoter of rDNA were reduced, both of which were also recovered by exogenous GFP-ING4 expression. Thus, ING4 could positively regulate rRNA transcription through modulation of histone modifications at the rDNA promoter.

A method for simultaneous targeted mutagenesis of all nuclear rDNA repeats in Saccharomyces cerevisiae using CRISPR-Cas9

Saccharomyces cerevisiae has been the prime model to study the assembly and functionality of eukaryotic ribosomes. Within that vast landscape, the specific problem of mutagenizing all 150 nuclear rRNA genes was bypassed using strains whose chromosomal copies had been deleted and substituted by plasmid-borne rDNA. Work with these strains has produced important insights, but nucleolar structure is altered and such yeast-specific approaches are elaborate and not transferable to most other eukaryotes. We describe here a simple CRISPR-Cas9 based method to place targeted mutations in all 150 chromosomal rDNA repeats in yeast. The procedure per se is not expected to alter the nucleolus and is potentially applicable also to other eukaryotes. Yeast was transformed with a plasmid bearing the genes for Cas9 and for the guide RNA, engineered to target a site in the SSU region. Our mutagenesis plan included insertion of a spliceosomal intron in the normally intronless yeast nuclear rDNA. Despite the potential lethality of cutting all 150 rDNA repeats at the same time, yeast survived the Cas9 attack through inactivation of the cut sites either by point mutations or by inserting the intron, which was spliced out correctly from the rRNA transcript. In each mutant strain the same mutation was present in all rDNA repeats and was stably inherited even after removal of the Cas9 plasmid.

TGFβ/Activin signalling is required for ribosome biogenesis and cell growth in Drosophila salivary glands

Signalling by TGFβ superfamily factors plays an important role in tissue growth and cell proliferation. In Drosophila , the activity of the TGFβ/Activin signalling branch has been linked to the regulation of cell growth and proliferation, but the cellular and molecular basis for these functions are not fully understood. In this study, we show that both the RII receptor Punt (Put) and the R-Smad Smad2 are strongly required for cell and tissue growth. Knocking down the expression of Put or Smad2 in salivary glands causes alterations in nucleolar structure and functions. Cells with decreased TGFβ/Activin signalling accumulate intermediate pre-rRNA transcripts containing internal transcribed spacer 1 regions accompanied by the nucleolar retention of ribosomal proteins. Thus, our results show that TGFβ/Activin signalling is required for ribosomal biogenesis, a key aspect of cellular growth control. Importantly, overexpression of Put enhanced cell growth induced by Drosophila Myc, a well-characterized inducer of nucleolar hypertrophy and ribosome biogenesis.