Non-Random Compositional Organization of Nuclear Speckles

Abstract

Eukaryotic nucleus is organized into discrete non-membranous sub-nuclear compartments. Studies on one such well-characterized domain, the nucleolus, revealed that its constituents are further compartmentalized into sub-nucleolar regions, and such organization is crucial for its functioning in ribosome biogenesis. However, presently it is not clear whether functional sub-compartmentalization is vital for the functioning of the large number of sub-nuclear domains present in the mammalian cell nucleus. Nuclear speckle is another sub-nuclear domain, which is enriched with protein and RNA components involved in mRNA metabolism, including transcription, pre-mRNA splicing and mRNA export. In the present study, by utilizing multi-color structured illumination microscopy and quantitative analysis, we determined the spatial organization of several components within the nuclear speckle, including mRNA transcripts, an lncRNA, constitutive splicing components, a splicing regulator, and a scaffold protein. Our data revealed a non-random organization of tested components, with constitutive splicing components defining a broader territory compared to splicing and scaffold protein. The nascent RNA transcripts from speckle-associated genes reside in different territories of the speckle in a gene-specific manner. Using highly transcribed speckle-associated Col1a1 gene as a specific example, we demonstrated that speckles containing the Col1a1 accumulated transcripts are in general are larger compared to the average speckles. Furthermore, blocking constitutive splicing and mRNA export affected Col1a1 transcript accumulation at the speckles, and correspondingly altered the transcription site-associated speckle size. Altogether, our studies revealed that the factors within nuclear speckles are arranged into sub-speckle territories, and such organization of speckles may be functionally important for their role in different steps of mRNA metabolism.