Proteomic Analysis of Interchromatin Granule Clusters

Abstract

A variety of proteins involved in gene expression have been localized within mammalian cell nuclei in a speckled distribution that predominantly corresponds to interchromatin granule clusters (IGCs). We have applied a mass spectrometry strategy to identify the protein composition of this nuclear organelle purified from mouse liver nuclei. Using this approach, we have identified 146 proteins, many of which had already been shown to be localized to IGCs, or their functions are common to other already identified IGC proteins. In addition, we identified 32 proteins for which only sequence information is available and thus these represent novel IGC protein candidates. We find that 54% of the identified IGC proteins have known functions in pre-mRNA splicing. In combination with proteins involved in other steps of pre-mRNA processing, 81% of the identified IGC proteins are associated with RNA metabolism. In addition, proteins involved in transcription, as well as several other cellular functions, have been identified in the IGC fraction. However, the predominance of pre-mRNA processing factors supports the proposed role of IGCs as assembly, modification, and/or storage sites for proteins involved in pre-mRNA processing.

Subnuclear Localization and Dynamics of the Pre-mRNA 3′ End Processing Factor Mammalian Cleavage Factor I 68-kDa Subunit

“…The RD/E dipeptide motif is similar to a phosphorylated RS domain, because the serine residue in the RS repeat is replaced with a negatively charged aspartic acid or glutamic acid residue. Consistent with this observation, the CF I m 68-kDa subunit was identified as a component of isolated IGCs (Saitoh et al, 2004).…”

“…The RD/E dipeptide motif is similar to a phosphorylated RS domain, because the serine residue in the RS repeat is replaced with a negatively charged aspartic acid or glutamic acid residue. Consistent with this observation, the CF I m 68-kDa subunit was identified as a component of isolated IGCs (Saitoh et al, 2004).We previously reported that all three CF I m subunits (25, 59, and 68 kDa) are nuclear at steady state. Specific antibodies detect the proteins in the nucleoplasm and in discrete foci (Dettwiler et al, 2004).…”

“…At the ultrastructural level, nuclear speckles can be divided into PFs, which contain nascent transcripts and are usually at the periphery of speckles, and IGCs. In contrast to the full-length protein, localization of the isolated RS-like domain was completely overlapping with nuclear speckles, possibly because of the lack of other regions of the protein Recently, 31 proteins with RS motifs, including CF I m 68, were identified in isolated IGCs (Saitoh et al, 2004). CF I m 68 belongs to a group of four of these proteins that is characterized by RE/RD domains.…”

“…In contrast to IGCs, PFs are labeled rapidly after short pulses with [ 3 H]uridine, suggesting that they represent nascent transcripts (for review, see Fakan, 1994). Recently, CF I m 68 was found in purified IGCs (Saitoh et al, 2004). Therefore, to study CF I m 68 localization at an ultrastructural level, immunoelectron microscopy was performed on HeLa cells with anti-CF I m 68 polyclonal antiserum.…”

Basonuclins 1 and 2, whose genes share a common origin, are proteins with widely different properties and functions

“…Many RNA processing factors, involved in either splicing or postsplicing processes such as mRNA export, nonsense-mediated decay, and polyadenylation, localize in speckles (16,(21)(22)(23)(24) and a speckled staining pattern indicates with near certainty a role in RNA processing (24)(25)(26). The virtually complete colocalization of bn2 with SC35 in nuclear speckles strongly suggests that bn2 has a function in RNA processing, and its unusual evolutionary stability implies that this function is essential.…”