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In mouse myoblasts nuclear prosomes are associated with the nuclear matrix and accumulate preferentially in the
F De Conto1, E Pilotti, S V Razin
1Institut Jacques Monod - Université Paris 7, Tour 43, France.
Abstract:
Prosomes are the core of 26S proteasomes, although they were originally observed as 20S particles associated with cytoplasmic mRNPs. Here we show for the first time that prosomes are also genuine constituents of the nuclear matrix, chromatin and the nuclear RNP networks. Using mouse myoblasts we tested three monoclonal antibodies recognising the prosomal subunits p23K, p27K and p30K, and found that the corresponding prosome subclasses are characterised by a variable distribution pattern within the nuclei. Their presence on the nuclear matrix, and most abundantly in the perinucleolar area, is of particular importance. When myoblasts fuse into myotubes, the distribution pattern of certain types of prosomes on the nuclear matrix changes drastically. Surprisingly, DNA strongly interferes with the detection of prosomal antigens by immunofluorescence methods, whereas RNA, histones and other proteins soluble in 2 M NaCl have no such effect. This 'masking' of prosomes can be completely overcome by extensive or even mild digestion with DNase I or restriction enzymes. Many nuclear prosomes can be solubilized by combined treatment with 0.5% Triton X-100 and 2 M NaCl, and others can be released by digestion of DNA and/or RNA, and about 10-20% of nuclear prosomes remain tightly bound to the protein-based nuclear matrix.
Insights
Prosomes, integral to proteasomes, are newly found within the cell nucleus, associated with chromatin and the nuclear matrix. Their nuclear presence and DNA interaction were revealed using specific antibodies and enzymatic treatments.
Area of Science:
- Cell Biology
- Molecular Biology
- Proteasome Research
Background:
- Prosomes, initially identified as 20S particles linked to cytoplasmic messenger ribonucleoprotein complexes (mRNPs), are core components of the 26S proteasome.
- Their presence and function within the cell nucleus have not been previously established.
Purpose of the Study:
- To investigate the presence and distribution of prosomes within the nuclear compartment.
- To explore the interaction of nuclear prosomes with DNA and other nuclear components.
- To characterize the dynamic changes in prosome localization during cellular differentiation.
Main Methods:
- Utilized three monoclonal antibodies targeting prosomal subunits (p23K, p27K, p30K) for immunofluorescence studies in mouse myoblasts.
- Employed DNase I and restriction enzymes to assess the role of DNA in prosome detection.
- Applied Triton X-100 and high salt concentrations for prosome solubilization.
Main Results:
- Demonstrated that prosomes are genuine constituents of the nuclear matrix, chromatin, and nuclear RNP networks.
- Observed variable distribution patterns of prosome subclasses within the nucleus, notably enriched in the perinucleolar area.
- Revealed that DNA significantly hinders immunofluorescence detection of prosomes, a 'masking' effect reversible by enzymatic digestion.
- Showcased dynamic redistribution of nuclear prosomes during myoblast fusion into myotubes.
Conclusions:
- Prosomes are not exclusively cytoplasmic but are integral nuclear components with specific localization patterns.
- Nuclear prosome detection is significantly influenced by DNA, suggesting a direct interaction or association.
- Prosome distribution undergoes significant alterations during cellular differentiation, indicating a role in nuclear remodeling.