The tripartite motif of nuclear factor 7 is required for its association with transcriptional units

Abstract

In amphibian oocytes, the maternal nuclear factor NF7 associates with the elongating pre-mRNAs present on the numerous lateral loops of the lampbrush chromosomes. Here, we have purified NF7 from an oocyte extract by using a combination of ion-exchange chromatography and gel filtration chromatography and demonstrated for the first time that nucleoplasmic NF7 exists primarily as free homotrimers. We confirmed the in vivo homotrimerization of NF7 by using a glutaraldehyde cross-linking assay, and we further showed that it only requires the coiled-coil domain of the NF7 tripartite motif/RBCC motif. Interestingly, we also obtained evidence that NF7 is recruited to the nucleus as a homotrimer, and expression of several mutated forms of NF7 in oocytes demonstrated that both the coiled coil and B box of NF7 are required for its chromosomal association. Together, these data strongly suggest that the interaction of NF7 with the active transcriptional units of RNA polymerase II is mediated by a trimeric B box. Finally, and in agreement with a role for NF7 in pre-mRNA maturation, we obtained evidence supporting the idea that NF7 associates with Cajal bodies.

FIG. 1.

NF7 is predicted to form trimers and associates with RNAPII active transcriptional units. (A) The Multicoil algorithm was used on Xenopus NF7 with a scanning window of 28 amino acid residues. A trimeric conformation was strongly predicted, with more than 80% probability for most of the coiled-coil region. This prediction is consistent with the data presented in Fig. 2 and 3, in which NF7 is demonstrated to form homotrimers. (B) Fluorescence micrograph showing a narrow region of an LBC labeled with anti-NF7 MAb 37-1A9 (green) and anti-XCAP-D2 (red). XCAP-D2, which is a subunit of condensin I, is restricted to the transcriptionally inactive heterochromatin domains of the LBCs (3) and was used here to define the chromosomal axes. In contrast, NF7 associates specifically with the RNP matrix of the chromosomal loops, which correspond to RNAPII active transcriptional units. In addition, NF7 is present on nucleoplasmic granules, which are out of focus here because of their small size (0.2 to 0.3 μm). Scale bar, 5 μm. (C) Western blot analysis with MAb 37-1A9 indicates that NF7 is primarily nuclear, and while it is found associated with chromosomes, it is also found soluble in the nucleoplasm. Proteins from 10 whole nuclei and organelles and nucleoplasm from 10 nuclei, five cytoplasms, or five whole oocytes were used. The values on the left are molecular sizes in kilodaltons.

FIG. 2.

Nucleoplasmic NF7 exists as a homotrimer. (A) Flow chart indicating the fractionation of nucleoplasmic NF7, which was followed through each step of purification by Western blot analysis with MAb 37-1A9. (B) Sypro orange-stained gel with each lane containing an equal volume of elution fractions 5 to 13 from the MonoS column. The major band with an apparent molecular mass of 80 kDa of fraction 09 was identified as NF7 by tandem mass spectrometry. The values on the left are molecular sizes in kilodaltons. (C) Fraction 09 from the MonoS column was further fractionated on a Sephacryl 200 column, and NF7 eluted at 240 kDa.

FIG. 3.

The coiled coil is essential for NF7 trimerization. (A) The cross-linking of endogenous NF7 trimers by 0.01% glutaraldehyde is demonstrated in the S-200 peak fractions and in a crude nucleoplasmic extract by Western blot analysis with MAb 37-1A9. Over time, a new band with an apparent molecular mass of ∼250 kDa is detected in addition to the monomeric NF7 form of 80 kDa. (B) Several modified forms of NF7 were produced in vitro in the presence of [³⁵S]methionine, and their ability to trimerize was tested by glutaraldehyde cross-linking. A schematic representation of the expressed proteins is given under each autoradiograph (R, RING finger; B, B box; CC, coiled coil; B30.2, RFP-like domain). Note that the only domain involved in multimerization is the coiled-coil region. The values on the left are molecular sizes in kilodaltons.

FIG. 4.

Trimerization and an intact B box are required for the association of NF7 with RNAPII active transcriptional units. (A) Transcripts of several modified forms of HA-tagged NF7 were injected into the cytoplasm of stage V oocytes, and nuclear spreads were prepared 18 h later. Newly made proteins (in red) were detected with anti-HA MAb 3F10 and an Alexa 594-conjugated secondary antibody. In all preparations, DNA was counterstained with picogreen (nucleoli and chromosomal axes are the only structures labeled) and the merged images are presented. A differential interference contrast image and its corresponding fluorescence micrograph are presented for ΔCC284-409 to emphasize the fact that while chromosomal loops are present, they are not labeled by MAb 3F10. Note that the two domains required for chromosomal association are the B box (ΔN275) and the coiled coil (ΔCC284-409). CBs, which were found to accumulate several of the newly expressed proteins, are indicated by arrows. Scale bars are10 μm. (B) Western blot analysis of the newly expressed protein with MAb 3F10. Each lane corresponds to 10 nuclei of stage V oocytes. The values on the left are molecular sizes in kilodaltons.

FIG. 5.

Association of NF7 with CBs. Differential interference contrast (DIC) and corresponding fluorescence micrographs of nuclear spreads. The DNA in both preparations was counterstained with picogreen. (A) The transcript coding for ΔCC284-409 was injected into the cytoplasm of stage V oocytes, and nuclear spreads were prepared 18 h later. The newly made protein was detected with MAb 3F10 (in red). Nucleoli, which are well labeled with picogreen, are weakly stained by MAb 3F10. In contrast, ΔCC284-409 is detected at a high concentration within CBs, as exemplified by the one present in the field (arrow). Note that the B snurposomes (arrowheads), which are the analogous structures of the somatic interchromatin granule clusters, are weakly labeled. (B) Immunostaining of endogenous NF7 with MAb 37-1A9 (in red). The lateral chromosomal loops and CBs (arrow) are well labeled, while nucleoli (green) and B snurposomes are negative. Scale bars are 5 μm.

FIG. 6.

NF7 trimerization occurs in the cytoplasm. Western blot assay showing the cellular distribution of two modified forms of NF7 upon expression in stage V oocytes. Both proteins were tagged with the HA epitope, and MAb 3F10 was used for their detection. Whether the transcripts coding for ΔNLS (80 kDa) and ΔC448 (60 kDa) were injected individually or coinjected is indicated by the plus and minus signs above the lanes. Cytoplasmic and nuclear protein samples were prepared 18 h after injection, and each lane corresponds to either five cytoplasms or 10 nuclei. The values on the left are molecular sizes in kilodaltons.

FIG. 7.

Paradigm of NF7 trimerization and association with chromosomal loops. In this model, NF7 trimerization mediated by the coiled-coil region occurs in the cytoplasm. NF7 homotrimers are then recruited to the nucleus, where they associate with the chromosomal loops through their trimeric B box. Whether NF7 transits through CBs remains uncertain.