Judith E. Sleeman

Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex

Many important cell mechanisms are carried out and regulated by multi-protein complexes, for example, transcription and RNA processing machinery, receptor complexes and cytoskeletal structures. Most of these complexes remain only partially characterized due to the difficulty of conventional protein analysis methods. The rapid expansion of DNA sequence databases now provides whole or partial gene sequences of model organisms, and recent advances in protein microcharacterization via mass spectrometry allow the possibility of linking these DNA sequences to the proteins in functional complexes. This approach has been demonstrated in organisms whose genomes have been sequenced, such as budding yeast. Here we report the first characterization of an entire mammalian multi-protein complex using these methods. The machinery that removes introns from mRNA precursors — the spliceosome — is a large multi-protein complex. Approximately half of the components excised from a two-dimensional gel separation of the spliceosome were found in protein sequence databases. Using nanoelectrospray mass spectrometry, the remainder were identified and cloned using public expressed sequence tag (EST) databases. Existing EST databases are thus already sufficiently complete to allow rapid characterization of large mammalian protein complexes via mass spectrometry.

Newly assembled snRNPs associate with coiled bodies before speckles, suggesting a nuclear snRNP maturation pathway

BACKGROUND Small nuclear ribonucleoproteins (snRNPs), which are essential components of the mRNA splicing machinery, comprise small nuclear RNAs, each complexed with a set of proteins. An early event in the maturation of snRNPs is the binding of the core proteins - the Sm proteins - to snRNAs in the cytoplasm followed by nuclear import. Immunolabelling with antibodies against Sm proteins shows that splicing snRNPs have a complex steady-state localisation within the nucleus, the result of the association of snRNPs with several distinct subnuclear structures. These include speckles, coiled bodies and nucleoli, in addition to a diffuse nucleoplasmic compartment. The reasons for snRNP accumulation in these different structures are unclear. RESULTS When mammalian cells were microinjected with plasmids encoding the Sm proteins B, D1 and E, each tagged with either the green fluorescent protein (GFP) or yellow-shifted GFP (YFP), a pulse of expression of the tagged proteins was observed. In each case, the newly synthesised GFP/YFP-labelled snRNPs accumulated first in coiled bodies and nucleoli, and later in nuclear speckles. Mature snRNPs localised immediately to speckles upon entering the nucleus after cell division. CONCLUSIONS The complex nuclear localisation of splicing snRNPs results, at least in part, from a specific pathway for newly assembled snRNPs. The data demonstrate that the distribution of snRNPs between coiled bodies and speckles is directed and not random.

Nuclear organization of pre-mRNA splicing factors.

The splicing of mRNA precursors (pre-mRNA) in the nucleus is catalyzed by a complex machinery termed the spliceosome. In order to understand how it functions in vivo, it is essential to complement biochemical analyses with a detailed study of how spliceosome components are organized within the nucleus.

Cajal body proteins SMN and Coilin show differential dynamic behaviour in vivo

Analysis of stable cell lines expressing fluorescently tagged survival of motor neurons protein (SMN) and coilin shows striking differences in their dynamic behaviour, both in the nucleus and during mitosis. Cajal bodies labelled with either FP-SMN or FP-coilin show similar behaviour and frequency of movements. However, fluorescence recovery after photobleaching (FRAP) studies show that SMN returns ∼50-fold more slowly to Cajal bodies than does coilin. Time-lapse studies on cells progressing from prophase through to G1 show further differences between SMN and coilin, both in their localisation in telophase and in the timing of their re-entry into daughter nuclei. The data reveal similarities between Cajal bodies and nucleoli in their behaviour during mitosis. This in vivo study indicates that SMN and coilin interact differentially with Cajal bodies and reveals parallels in the pathway for reassembly of nucleoli and Cajal bodies following mitosis.

Nuclear substructure and dynamics

Many aspects of nuclear organization exhibit dynamic properties. Both nuclear bodies and individual chromatin regions can move through the nucleoplasm. Certain nuclear bodies are able to assemble and disassemble from soluble components in response to the metabolic demands of the cell. The cycling behaviour of nuclear factors and the rapid turnover of nuclear body components also underlines the importance of measuring the dynamic properties of proteins and RNPs when interpreting both localization patterns and their possible functional roles. Static fluorescence or electron microscopy images usually indicate steady state accumulations of nuclear factors, but can fail to reveal sites where factors localize transiently. Recent work has also shown that some nuclear proteins and RNPs participate in directional trafficking pathways, moving in a temporal sequence between distinct classes of nuclear bodies. For example, the nucleolar snoRNP protein NHPX/p15.5kDa transiently accumulates in splicing speckles before adopting a steady state concentration in nucleoli and Cajal bodies.Our understanding of the dynamic organization of the cell nucleus is expanding rapidly and this is facilitated by the increasing range of new methods to study the composition, structure and movement of nuclear bodies and their components. In the case of nucleoli, NPCs, speckles and Cajal bodies, they can be purified and their protein components can be identified using mass spectrometry. The ability to label nuclear structures in vivo through the expression of fluorescent protein-tagged factors also now allows many dynamic parameters to be measured directly in living cells using photobleaching techniques. Further developments in quantitative fluorescence microscopy and quantitative proteomics will help to develop a more robust model of how the nucleus is organized and should inform our view as to how nuclear structure influences cellular function.

A regulatory role for CRM1 in the multi-directional trafficking of splicing snRNPs in the mammalian nucleus

Distinct pathways of ribonucleoprotein transport exist within the nucleus, connected to their biogenesis and maturation. These occur despite evidence that the major mechanism for their movement within the nucleus is passive diffusion. Using fusions of Sm proteins to YFP, CFP and photoactivatable GFP, I have demonstrated that pathways with uni-directional bulk flow of complexes can be maintained within the nucleus despite multi-directional exchange of individual complexes. Newly imported splicing small nuclear ribonucleoproteins (snRNPs) exchange between Cajal bodies (CBs) within a nucleus and access the cytoplasm, but are unable to accumulate in speckles. By contrast, snRNPs at steady-state exchange freely in any direction between CBs and speckles, but cannot leave the nucleus. In addition to these surprising qualitative observations in the behaviour of nuclear complexes, sensitive live-cell microscopy techniques can detect subtle quantitative disturbances in nuclear dynamics before they have had an effect on overall nuclear organization. Inhibition of the nuclear export factor, CRM1, using leptomycin B results in a change in the dynamics of interaction of newly imported snRNPs with CBs. Together with the detection of interactions of CRM1 with Sm proteins and the survival of motor neurons (SMN) protein, these studies suggest that the export receptor CRM1 is a key player in the molecular mechanism for maintaining these pathways. Its role in snRNP trafficking, however, appears to be distinct from its previously identified role in small nucleolar RNP (snoRNP) maturation.

Molecular and functional characterization of microsomal UDP-glucuronic acid uptake by members of the nucleotide sugar transporter (NST) family

Transport of the co-substrate UDPGA (UDP-glucuronic acid) into the lumen of the endoplasmic reticulum is an essential step in glucuronidation reactions due to the intraluminal location of the catalytic site of the enzyme UGT (UDP-glucuronosyltransferase). In the present study, we have characterized the function of several NSTs (nucleotide sugar transporters) and UGTs as potential carriers of UDPGA for glucuronidation reactions. UDPGlcNAc (UDP-N-acetylglucosamine)-dependent UDPGA uptake was found both in rat liver microsomes and in microsomes prepared from the rat hepatoma cell line H4IIE. The latency of UGT activity in microsomes derived from rat liver and V79 cells expressing UGT1A6 correlated well with mannose-6-phosphatase latency, confirming the UGT in the recombinant cells retained a physiology similar to rat liver microsomes. In the present study, four cDNAs coding for NSTs were obtained; two were previously reported (UGTrel1 and UGTrel7) and two newly identified (huYEA4 and huYEA4S). Localization of NSTs within the human genome sequence revealed that huYEA4S is an alternatively spliced form of huYEA4. All the cloned NSTs were stably expressed in V79 (Chinese hamster fibroblast) cells, and were able to transport UDPGA after preloading of isolated microsomal vesicles with UDPGlcNAc. The highest uptake was seen with UGTrel7, which displayed a V(max) approx. 1% of rat liver microsomes. Treatment of H4IIE cells with beta-naphthoflavone induced UGT protein expression but did not affect the rate of UDPGA uptake. Furthermore, microsomes from UGT1-deficient Gunn rat liver showed UDPGA uptake similar to those from control rats. These data show that NSTs can act as UDPGA transporters for glucuronidation reactions, and indicate that UGTs of the 1A family do not function as UDPGA carriers in microsomes. The cell line H4IIE is a useful model for the study of UDPGA transporters for glucuronidation reactions.

The SMN Protein is a Key Regulator of Nuclear Architecture in Differentiating Neuroblastoma Cells

The cell nucleus contains two closely related structures, Cajal bodies (CBs) and gems. CBs are the first site of accumulation of newly assembled splicing snRNPs (small nuclear ribonucleoproteins) following their import into the nucleus, before they form their steady‐state localization in nuclear splicing speckles. Gems are the nuclear site of accumulation of survival motor neurons (SMNs), an insufficiency of which leads to the inherited neurodegenerative condition, spinal muscular atrophy (SMA). SMN is required in the cytoplasm for the addition of core, Sm, proteins to new snRNPs and is believed to accompany snRNPs to the CB. In most cell lines, gems are indistinguishable from CBs, although the structures are often separate in vivo. The relationship between CBs and gems is not fully understood, but there is evidence that symmetrical dimethylation of arginine residues in the CB protein coilin brings them together in HeLa cells. During neuronal differentiation of the human neuroblastoma cell line SH‐SY5Y, CBs and gems increase their colocalization, mimicking changes seen during foetal development. This does not result from alterations in the methylation of coilin, but from increased levels of SMN. Expression of exogenous SMN results in an increased efficiency of snRNP transport to nuclear speckles. This suggests different mechanisms are present in different cell types and in vivo that may be significant for the tissue‐specific pathology of SMA.

Small nuclear RNAs and mRNAs: linking RNA processing and transport to spinal muscular atrophy

The splicing of pre-mRNA by the spliceosome is a characteristic feature of eukaryotic cells, dependent on a group of snRNPs (small nuclear ribonucleoproteins). These splicing snRNPs have a complex assembly pathway involving multiple steps that take place in different regions of the cell, which is reflected in their complex subcellular distribution. Vital to the assembly of splicing snRNPs is the protein SMN (survival of motor neurons). In multicellular organisms, SMN acts in the cytoplasm, together with its associated protein complex to assemble a heptameric ring of proteins called the Sm proteins as an early stage in splicing snRNP assembly. A deficiency of the SMN protein results in the inherited neurodegenerative condition SMA (spinal muscular atrophy), a leading cause of infant mortality specifically affecting spinal motor neurons. It has long been a puzzle how lowered levels of a protein required for a process as fundamental as splicing snRNP assembly can result in a condition with such a definite cell-type-specificity. The present review highlights recent research that points to wider roles in RNA metabolism for both SMN itself and the Sm proteins with which it is linked.

The Cajal body and the nucleolus: “In a relationship” or “It's complicated”?

ABSTRACT From their initial identification as ‘nucleolar accessory bodies’ more than a century ago, the relationship between Cajal bodies and nucleoli has been a subject of interest and controversy. In this review, we seek to place recent developments in the understanding of the physical and functional relationships between the 2 structures in the context of historical observations. Biophysical models of nuclear body formation, the molecular nature of CB/nucleolus interactions and the increasing list of joint roles for CBs and nucleoli, predominantly in assembling ribonucleoprotein (RNP) complexes, are discussed.