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.

Figure 1.

Profile of the Identified IGC proteins. One hundred forty-six identified IGC proteins are categorized based upon their proposed functions; 81% of the proteins are involved in activities related to RNA metabolism.

Figure 2.

In vivo localization of several novel IGC proteins. The cDNAs for several novel IGC protein candidates (KIAA0164 = Btf, KIAA0670 = acinus, KIAA0801 = RNA helicase, KIAA1019 = son protein) were fused to yellow fluorescent protein and expressed in A431 cells. The cells were fixed and labeled with an antibody to the pre-mRNA splicing factor SC35 (Fu and Maniatis, 1990), which localizes in IGCs.

Figure 3.

RS domain-containing proteins detected in the IGCs. Thirty-one proteins with RS motifs were detected in the IGC fraction and were categorized into three subgroups. Proteins in the first group (A) are of relatively low molecular mass, contain one or more RRMs, and many are founding members of the SR protein family. Proteins in the second group (B) are of larger molecular mass, and most do not contain an RRM but do contain additional motifs. Proteins in the third group (C) are also of higher molecular mass and contain repetitive sequences.