The A-type lamins have been observed to colocalize with RNA splicing factors in speckles within the nucleus, in addition to their typical distribution at the nuclear periphery. To understand the functions of lamin speckles, the effects of transcriptional inhibitors known to modify RNA splicing factor compartments (SFCs) were examined. Treatment of HeLa cells with α-amanitin or 5,6-dichlorobenzimidazole riboside (DRB) inhibited RNA polymerase II (pol II) transcription and led to the enlargement of lamin speckles as well as SFCs. Removal of the reversible inhibitor DRB resulted in the reactivation of transcription and a rapid, synchronous redistribution of lamins and splicing factors to normal-sized speckles, indicating a close association between lamin speckles and SFCs. Conversely, the expression of NH2-terminally modified lamin A or C in HeLa cells brought about a loss of lamin speckles, depletion of SFCs, and down-regulation of pol II transcription without affecting the peripheral lamina. Our results suggest a unique role for lamin speckles in the spatial organization of RNA splicing factors and pol II transcription in the nucleus.

Lamins are nuclear proteins that are components of a fibrous network underlying the inner nuclear membrane termed the nuclear lamina, and are also distributed throughout the interior of the nucleus (reviewed by Goldman et al., 2002). Lamins have been classified into two types based on biochemical properties and expression patterns. B-type lamins are expressed in most cells and are encoded by two separate genes, B1 and B2, whereas the A-type lamins have been detected primarily in differentiated cell types. The lamin A gene (LMNA) encodes lamin A and C transcripts, as well as germcell-specific lamin C2. Lamins belong to the intermediate filament family of proteins and have a short N-terminal head domain followed by an ␣-helical rod domain and a globular tail domain. The C-termini of lamins A, B1 and B2 bear a CAAX motif that is post-translationally modified and, in the case of lamin A, is subjected to a further maturation step by which prelamin A is proteolytically cleaved to give mature lamin A (reviewed by Stuurman et al., 1998).Mutations in human LMNA cause several debilitating diseases, collectively termed laminopathies, that affect skeletal and cardiac muscle, adipose, bone and neuronal tissues, and also cause premature ageing syndromes. The majority of mutations cause autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) (Bonne et al., 1999), whereas other mutations cause dilated cardiomyopathy with conduction system disease (DCM) (Fatkin et al., 1999), limb girdle muscular dystrophy , or familial partial lipodystrophy (FPLD) (Shackleton et al., 2000;Cao and Hegele, 2000). Mutations in LMNA have also been linked to the relatively rare autosomal recessive disorders CharcotMarie-Tooth disorder type 2 (De Sandre-Giovannoli et al., 2002) and mandibuloacral dysplasia (Novelli et al., 2002), and more recently to restrictive dermopathy (Navarro et al., 2004). Many of the above mutations are missense mutations that occur throughout the gene, although the FPLD mutations are clustered near the C-terminus. An interesting finding has been the linkage of mutations in LMNA to Hutchinson-Gilford progeria syndrome (HGPS) (Eriksson et al., 2003; De SandreGiovannoli et al., 2003) and to atypical Werner's syndrome . The most frequent mutation in HGPS is a nucleotide substitution (GGC to GGT) that does not cause an amino acid change (G608G) but activates a cryptic splice site that leads to a deletion of 50 amino acids near the C-terminus (residues 607-656), giving rise to a truncated protein termed lamin A del50 or progerin, which retains the C-terminus of prelamin A and is not proteolytically processed. A few missense mutations such as R471C and R527C that do not affect processing of the C-terminus of lamin A have also been linked to HGPS and atypical progerias (Eriksson et al., 2003;Cao and Hegele, 2003). Cells from patients expressing mutant lamins often exhibit a range of dominant-negative effects such as abnormal nuclear morphology, aberrant lamin assembly and altered gene regulation (reviewed by Wilson, 2000;Worman...

Lamins are the major structural components of the nucleus and mutations in the human lamin A gene cause a number of genetic diseases collectively termed laminopathies. At the cellular level, lamin A mutations cause aberrant nuclear morphology and defects in nuclear functions such as the response to DNA damage. We have investigated the mechanism of depletion of a key damage sensor, ATR (Ataxia-telangiectasia-mutated-and-Rad3-related) kinase, in HeLa cells expressing lamin A mutants or lamin A shRNA. The degradation of ATR kinase in these cells was through the proteasomal pathway as it was reversed by the proteasomal inhibitor MG132. Expression of lamin A mutants or shRNA led to transcriptional activation of three ubiquitin ligase components, namely, RNF123 (ring finger protein 123), HECW2 (HECT domain ligase W2) and the F-box protein FBXW10. Ectopic expression of RNF123, HECW2 or FBXW10 directly resulted in proteasomal degradation of ATR kinase and the ring domain of RNF123 was required for this degradation. However, these ligases did not alter the stability of DNA-dependent protein kinase, which is not depleted upon lamin misexpression. Although degradation of ATR kinase was reversed by MG132, it was not affected by the nuclear export inhibitor, leptomycin B, suggesting that ATR kinase is degraded within the nucleus. Our findings indicate that lamin misexpression can lead to deleterious effects on the stability of the key DNA damage sensor, ATR kinase by upregulation of specific components of the ubiquitination pathway.

The first example of a Ras family GTPase and its exchange factor C3G localizing to nuclear speckles and regulating mRNA splicing is presented.

Summary: Nuclear speckles are sites for pre-mRNA splicing. We provide evidence for localization and function of a Ras family GTPase, Rap1 and its exchange factor C3G in nuclear speckles.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/159269 doi: bioRxiv preprint first posted online 2 Abstract: C3G (RapGEF1), essential for mammalian embryonic development, is ubiquitously expressed and undergoes regulated nucleo-cytoplasmic exchange. Here we show that C3G localizes to SC35 positive nuclear speckles, and regulates splicing activity. Reversible association of C3G with speckles was seen upon inhibition of transcription and splicing. C3Gshows partial colocalization with SC35, and is recruited to a chromatin and RNase sensitive fraction of speckles. Its presence in speckles is dependent on intact cellular actin cytoskeleton, and is lost upon expression of the kinase, Clk1. Rap1, a substrate of C3G, is also present in nuclear speckles and inactivation of Rap signalling by expression of GFPRap1GAP, alters speckle morphology and number. Enhanced association of C3G with speckles is seen upon GSK3β inhibition, or differentiation of C2C12 cells to myotubes.CRISPR/Cas9 mediated knockdown of C3G resulted in decreased splicing activity and reduced staining for SC35 in speckles. C3G knockout clones of C2C12 as well as MDA-MB-231 showed reduced protein levels of several splicing factors compared to control cells. Our results identify C3G and Rap1 as novel components of nuclear speckles and a role for C3G in regulating cellular RNA splicing activity.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.