Research Focus Compartmentalization of the splicing machinery in plant cell nuclei Zdravko J. Lorkovic ´ and Andrea Barta Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, Department of Biochemistry, Medical University of Vienna, Dr. Bohrgasse 9/3, A-1030 Vienna, Austria The cell nucleus is a membrane-surrounded organelle that contains numerous compartments in addition to chromatin. Compartmentalization of the nucleus is now accepted as an important feature for the organization of nuclear processes and for gene expression. Recent studies on nuclear organization of splicing factors in plant cells provide insights into the compartmentaliza- tion of the plant cell nuclei and conservation of nuclear compartments between plants and metazoans. Most aspects of eukaryotic gene expression take place in the nucleus, a multifunctional cellular organelle. The multi- functionality of the nucleus is also reflected in its morpho- logical complexity. In addition to the nucleolus (Box 1), a still increasing number of different nuclear compartments have been described in vertebrate nuclei [1,2]. Of these, the most prominent are speckles (Box 1), in which the majority of splicing factors are localized [1,3], and Cajal bodies (Box 1), which in addition to spliceosomal small nuclear ribonucleoprotein particles (snRNPs) (Box 2) contain components involved in transcription by RNA polymerase I, II and III and those involved in modifying rRNA and small nuclear (sn)RNAs [4–6]. Although they are often referred to as nuclear organelles, nuclear compart- ments are not separated from each other by membranes. However, the protein composition of each nuclear compart- ment seems to be highly specific. Furthermore, they can be morphologically identified by electron and fluorescent microscopy, and at least some of the nuclear compartments can be biochemically purified. In addition, some of the nuclear compartments are highly dynamic structures, as evident by the continuous exchange of protein and RNA-protein components between different domains [1–7]. The complexity of the plant cell nucleus is just beginning to emerge and so far not much is known about the compartmentalization of RNA-processing machineries in plant cells. Different approaches have been used to demonstrate that spliceosomal components in plant nuclei are localized in a diffuse nucleoplasmic network and in Cajal bodies [8,9]. By using fluorescent protein fusion technology in combination with confocal as well as electron microscopy, recent studies from several groups have clearly demonstrated that plant nuclei also contain speckles [10–14], which are dynamic structures like their mammalian counterparts [11–13]. Speckles in plant cell nuclei? Although early studies with an antibody against the U2 snRNP-specific protein U2B 00 revealed that splicing factors in plant nuclei localize to Cajal bodies and also to a diffuse nucleoplasmic network [8], the existence of Box 1. Nuclear compartments Nucleolus This is the most prominent nuclear structure, clearly visible by bright-field microscopy. The nucleolus is the site of ribosomal RNA (rRNA) synthesis, processing and modification, and for the assembly of ribosomal subunits. It is organized into distinct regions, which in plant and animal cells reflect the step-wise process of ribosomal biogenesis. More recently, a variety of other putative functions such as biogenesis and transport of (m)RNAs and ribonucleoprotein particles (RNPs), cell cycle control and stress responses have been suggested for the nucleolus [27,29,30]. Speckle In metazoan nuclei, speckles have been defined as compartments of variable size (0.5–1.8 mm) and irregular shape, seen as interchromatin granule clusters by electron microscopy. They are also called ‘SC35 domain’ or ‘splicing factor compartment’ because splicing factors are highly enriched in them. However, they are not primary sites of pre-mRNA splicing because most active genes are found at the periphery of speckles. It seems that speckles serve as storage or assembly sites of spliceosomal components. They are dynamic structures, and proteins and ribonucleoprotein complexes are recruited from them to sites of transcription [31]. Although a speckled localization pattern is highly diagnostic for proteins involved in splicing, some other proteins have been found in these domains, including some transcription factors, RNA polymerase II subunits, RNA 3 0 -end processing factors and lamin A. Also, poly(A) C RNA has been found in speckles to some extent [1,3,28]. Cajal body Cajal bodies (also known as coiled bodies), are spherical structures ranging in size from 0.2–1.0 mm (up to 2.0 mm in plant cells) and localized in the nucleoplasm or close to the nucleolus. In plant nuclei, the majority of Cajal bodies are closely associated with nucleoli. Cajal bodies move within the nucleus in plant and animal cells. The function of Cajal bodies is not clear but recent experiments indicate multiple functions, including assembly of transcriptomes, small nuclear RNP assembly, modification of spliceosomal small nuclear RNAs and trafficking of small nucleolar RNPs involved in modifying rRNA in the nucleolus [4–6]. Transcription sites They appear as several thousands foci throughout the nucleoplasm of plant and animal cells and they are often localized on the periphery of interchromatin granule clusters or speckles. RNA polymerase II components co-localize with these sites. Although pre-mRNA splicing occurs co-transcriptionally, splicing factors do not strongly accumulate at these sites. Corresponding author: Zdravko J. Lorkovic ´ (zdravko.lorkovic@univie.ac.at or zdravko.lorkovic@meduniwien.ac.at). Available online 5 November 2004 Update TRENDS in Plant Science Vol.9 No.12 December 2004 www.sciencedirect.com