The DNA-Binding and τ2 Transactivation Domains of the Rat Glucocorticoid Receptor Constitute a Nuclear Matrix-Targeting Signal

The Ser-Arg (SR)-related protein SRm160 is a coactivator of premRNA splicing. It bridges splicing factors located at the 5 splice site, branch site, and 3 splice site. Recently, SRm160 has also been shown to be involved in mRNA export as part of an exon-junction complex. SRm160 is highly concentrated in splicing speckles but is also present in long branched intranuclear tracks connecting splicing speckles with sites at the nuclear lamina. In this study we identified domains of SRm160 important for spatial targeting within the nucleus and for binding to the nuclear matrix. Using a series of FLAG-and enhanced GFP-conjugated deletion mutants we found two contiguous sequences that independently target SRm160 to nuclear matrix sites at splicing speckled domains: amino acids 300 -350 and 351-688. Constructs containing amino acids 300 -350 were also targeted to sites peripheral to speckled domains where most mRNA originate subsequent to splicing. Sequences from the N-terminal domain localized proteins to the nuclear lamina near sites where mRNA leaves the nucleus.RNA splicing ͉ RNA export ͉ speckled domains ͉ SRm300 S ince the discovery of RNA splicing (1), its mechanism has been elucidated by the clever use of in vitro assays (2). Simple precursor RNAs, usually with one small intron, are added to a nuclear extract. After the addition of ATP, spliceosomal complexes form, and introns are removed slowly. In marked contrast, native RNA splicing in cells is far more rapid and efficient, capable of processing more complicated substrates. Precursor RNAs as large as 80,780 bases with as many as 175 introns (3) are rapidly spliced, often in complicated but precise alternative patterns. The very rapid splicing seen in vivo likely reflects, in part, the accurate positioning of splicing substrates and factors by the highly ordered architecture of the nucleus. Most RNA splicing factors are concentrated in subnuclear structures that appear as speckled domains when visualized by immunofluorescence microscopy (4). When seen by electron microscopy, these correspond to interchromatin granule clusters (5) that are surrounded by regions rich in the perichromatin fibrils that contain many new transcripts (5, 6). A majority of these transcripts are spliced at or near speckled domains (7), and mechanisms have been described for recruiting splicing factors from these domains to newly activated genes (8,9).Evidence that the nuclear matrix has a critical role in RNA splicing has emerged from studies examining cells expressing a ␤-globin pre-mRNA splicing construct (10,11). This precursor remains associated with the nuclear matrix after its isolation and is spliced rapidly after addition of the ATP (11). In contrast to conventional in vitro splicing reactions, splicing in situ on nuclear matrix preparations occurs without a lag period, indicating that spliceosomal commitment complexes are preassembled and fully functional.Two strong candidates for factors that might couple splicing components are Ser-Arg (SR)-related matrix protein of 160 kDa (...

Section: Discussionmentioning

confidence: 99%

The spatial targeting and nuclear matrix binding domains of SRm160

Wagner

Chiosea

Nickerson

2003

“…The NMTS directs this hematopoietic transcription factor to nuclear matrix-associated foci that support gene expression (35). Other classes of gene regulatory factors, including the androgen and glucocorticoid receptors (52,53), Pit-1 (54), and YY-1 (50,55), have also been shown to contain NMTSs that differ from the AML1 NMTS. This diversity of trafficking signals may provide the requisite specificity to direct factors to independent subnuclear locations.…”

Section: Discussionmentioning

confidence: 99%

The t(8;21) chromosomal translocation in acute myelogenous leukemia modifies intranuclear targeting of the AML1/CBFα2 transcription factor

McNeil

Zeng

Harrington

et al. 1999

Targeting of gene regulatory factors to specific intranuclear sites may be critical for the accurate control of gene expression. The acute myelogenous leukemia 8;21 (AML1͞ETO) fusion protein is encoded by a rearranged gene created by the ETO chromosomal translocation. This protein lacks the nuclear matrix-targeting signal that directs the AML1 protein to appropriate gene regulatory sites within the nucleus. Here we report that substitution of the chromosome 8-derived ETO protein for the multifunctional C terminus of AML1 precludes targeting of the factor to AML1 subnuclear domains. Instead, the AML1͞ETO fusion protein is redirected by the ETO component to alternate nuclear matrix-associated foci. Our results link the ETO chromosomal translocation in AML with modifications in the intranuclear trafficking of the key hematopoietic regulatory factor, AML1. We conclude that misrouting of gene regulatory factors as a consequence of chromosomal translocations is an important characteristic of acute leukemias. M odifications in nuclear morphology are a hallmark of tumor cells and are used for diagnosis. However, there is limited knowledge of the subnuclear changes that accompany or contribute to tumor-related alterations of nuclear architecture. Biochemical evidence indicates that the protein composition of the nuclear matrix is modified in tumor cells (1-7). The nuclear matrix is a scaffold that provides a means for localizing genes and regulatory factors throughout the nuclear space (8-11). Consequently, analysis of targeting signals that direct regulatory factors to nuclear matrix-associated subnuclear sites (12) may provide insight into nuclear structure-function relationships that are compromised in cancer.The acute myelogenous leukemia (AML) transcription factor AML1 1 is a key regulator of hematopoiesis (13,14). Numerous cytogenetic abnormalities that involve genes encoding AML1 or its partner core binding factor ␤ have been identified in AML and acute lymphocytic leukemia (15-18). The frequent 8;21 translocation produces a chimeric protein (AML1͞ETO) in which the C terminus of AML1 is replaced by the unrelated ETO (MTG8) protein (19-23). The 8;21 translocation occurs in approximately 15% of AML in adult patients (24-26).The normal form of the AML1 protein has an N-terminal region containing a runt homology DNA-binding domain (27) and a multifunctional C-terminal region that supports transactivation, repression, and intranuclear targeting (28-34). Alternative splicing of the AML1 gene can generate several protein isoforms. The predominant isoform AML1B (480 aa) contains a 31-aa nuclear matrix-targeting signal (NMTS) in the C terminus. This targeting signal is necessary and sufficient to direct the factor to nuclear matrix-associated subnuclear sites that support transcription (30). Many of these AML1 sites are localized together with the hyperphosphorylated active form of RNA polymerase II o , and this association requires the presence of the runt homology domain of AML1B (35).The AML1͞ETO fusion product is expre...

“…F actors that mediate transcription, processing of gene transcripts, DNA replication, and DNA repair are organized as discrete domains within the nucleus (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). The osteogenic and hematopoietic Runx transcription factors contain a unique and conserved amino acid motif in the C terminus designated the nuclear matrix targeting signal that directs Runx proteins to subnuclear foci where gene regulatory complexes are assembled in situ (14)(15)(16)(17)(18)(19)(20).…”

mentioning

confidence: 99%

Subnuclear targeting of Runx/Cbfa/AML factors is essential for tissue-specific differentiation during embryonic development

Choi

Pratap

Javed

et al. 2001

256

279

Runx (Cbfa͞AML) transcription factors are critical for tissue-specific gene expression. A unique targeting signal in the C terminus directs Runx factors to discrete foci within the nucleus. Using Runx2͞CBFA1͞AML3 and its essential role in osteogenesis as a model, we investigated the fundamental importance of fidelity of subnuclear localization for tissue differentiating activity by deleting the intranuclear targeting signal via homologous recombination. Mice homozygous for the deletion (Runx2⌬C) do not form bone due to maturational arrest of osteoblasts. Heterozygotes do not develop clavicles, but are otherwise normal. These phenotypes are indistinguishable from those of the homozygous and heterozygous null mutants, indicating that the intranuclear targeting signal is a critical determinant for function. The expressed truncated Runx2⌬C protein enters the nucleus and retains normal DNA binding activity, but shows complete loss of intranuclear targeting. These results demonstrate that the multifunctional N-terminal region of the Runx2 protein is not sufficient for biological activity. We conclude that subnuclear localization of Runx factors in specific foci together with associated regulatory functions is essential for control of Runx-dependent genes involved in tissue differentiation during embryonic development.F actors that mediate transcription, processing of gene transcripts, DNA replication, and DNA repair are organized as discrete domains within the nucleus (1-13). The osteogenic and hematopoietic Runx transcription factors contain a unique and conserved amino acid motif in the C terminus designated the nuclear matrix targeting signal that directs Runx proteins to subnuclear foci where gene regulatory complexes are assembled in situ (14)(15)(16)(17)(18)(19)(20). Thus the Runx transcription factors provide a paradigm for pursuing mechanisms that coordinate the compartmentalization of regulatory proteins in intranuclear sites. In this study we directly addressed the fundamental question of whether subnuclear targeting and the associated regulatory activities of Runx factors are obligatory for in vivo function.The Runx (CBFA͞AML͞PEBP2␣) ʈ family of transcription factors are critical for cellular differentiation and organ development (21-23). Key studies have established that Runx2͞ CBFA1͞AML3 is required for osteoblast differentiation and in vivo bone formation (24-28). Ablation of the Runx2 gene in mice results in a complete absence of intramembranous and endochondral bone that is attributed to the maturational arrest of hypertrophic chondrocytes and osteoblasts (26,29,30). Haploinsufficiency of this gene results in the human syndrome cleidocranial dysplasia, a dominantly inherited developmental disorder of bone (24,25). A basis is thereby provided for examining the biological consequences resulting from absence of the Cterminal targeting function.Runx factors share multiple functional domains. The Nterminal runt homology DNA binding domain (RHD), which interacts with many coregulatory factors and chromatin m...