REF1/Aly and the additional exon junction complex proteins are dispensable for nuclear mRNA export

Abstract

The metazoan proteins UAP56, REF1, and NXF1 are thought to bind sequentially to mRNA to promote its export to the cytoplasm: UAP56 is thought to recruit REF1 to nascent mRNA; REF1 acts as an adaptor protein mediating the association of NXF1 with mRNA, whereas NXF1 translocates the mRNA across the nuclear pore complex. REF1 is a component of the exon-exon junction complex (EJC); thus, the EJC is thought to play a role in the export of spliced mRNA. NXF1 and UAP56 are essential for mRNA export. An essential role for metazoan REF1 or the additional EJC proteins in this process has not been established. Contrary to expectation, we show that REF1 and the additional components of the EJC are dispensable for export of bulk mRNA in Drosophila cells. Only when REF1 and RNPS1 are codepleted, or when all EJC proteins are simultaneously depleted is a partial nuclear accumulation of polyadenylated RNAs observed. Because a significant fraction of bulk mRNA is detected in the cytoplasm of cells depleted of all EJC proteins, we conclude that additional adaptor protein(s) mediate the interaction between NXF1 and cellular mRNAs in metazoa. Our results imply that the essential role of UAP56 in mRNA export is not restricted to the recruitment of REF1.

Figure 1.

Expression and domain organization of Drosophila EJC proteins. (A) Schematic sequence relationships among Drosophila and vertebrate components of the EJC. The numbers in italics indicate the length of the amino acid sequence. The sequence identities between Drosophila and human proteins are indicated on the right. PWI, a motif characterized by a conserved PWI sequence (Blencowe and Ouzounis, 1999); RBD, RNP-type RNA binding domain (the RNP1 and RNP2 motifs are shown in red); SAP, a putative DNA binding motif (Aravind and Koonin, 2000); SR-domain, Serine and Arginine rich domain. The REF proteins are characterized by the presence of two conserved motifs at their NH₂ and COOH termini known as REF-N (N) and REF-C (C) boxes (Stutz et al., 2000). The REF-C box is missing in Dm REF2. RNPS1 is characterized by an RBD and an NH₂-terminal sequence rich in serines (S-rich). Despite sequence similarities and a similar domain organization, Dm DEK is almost twice the size of human DEK. (B) Detection of Dm dek, ref1, ref2, rnps1, srm160, and y14 mRNAs by Northern blot analysis. 25 μg of total RNA from SL2 cells was loaded per lane. For ref2, no signal was detected even after prolonged exposures. (C) SL2 cells were transfected with dsRNA specific for Dm REF1 (lane 4), Dm REF2 (lane 6), or a mixture of both dsRNAs (lane 5). Protein samples from total lysates of untreated or depleted cells were analyzed by Western blot using two anti-REF antisera (KJ58 and KJ70). 1 μl aliquots of unlabeled Dm REF1 or Dm REF2 synthesized in vitro in E. coli lysates were analyzed in parallel (lanes 1 and 2).

Figure 2.

Drosophila cells depleted of EJC proteins display diverse growth phenotypes. (A) SL2 cells growing in suspension were transfected with dsRNAs specific for DEK, NXF1, REF1, REF2, RNPS1, SRm160, and Y14. Cell numbers were determined every day up to 12 d after transfection. On day 7, cells were retransfected with the corresponding dsRNAs, with the exception of NXF1-depleted cells. Data are given as the n-fold increase in cell numbers relative to the amount of cells used for transfection on day 0 (where n represents a number on the y axis). Note that on the y axis the scale is logarithmic. (B) Aliquots of cells shown in panel A were collected on days 4 and 11 and analyzed by Western blotting with antibodies raised against the recombinant proteins. The steady-state expression level of targeted proteins was reduced, whereas the expression level of the unrelated protein tubulin is not affected (lanes 5–7). In lanes 1–4, dilutions of the samples isolated on day 0 were loaded to analyze the efficiency of the depletion. (C) SL2 cells were transfected with dsRNAs specific for DEK, RNPS1, SRm160, and Y14 as indicated above the panels. 11 d after transfection cell lysates were analyzed by Western blotting with antibodies specific for the corresponding proteins. The expression level of REF1 is shown in the bottom panels.

Figure 3.

REF1/Aly is dispensable for mRNA export in Drosophila cells. SL2 cells treated with GFP (control), REF1, or REF2 dsRNAs were fixed 11 d after transfection and stained with antibodies that specifically recognize Dm REF1 (KJ58). Cells were double labeled by FISH with a Cy3-labeled oligo(dT) probe (poly[A]⁺ RNA). The arrowheads point to cells in which a nuclear accumulation of poly(A)⁺ RNA was observed. These represent <20% of the cell population. The arrows point to cells in which the nucleus is not in the focal plane.

Figure 4.

Depletion of EJC proteins reduces, but does not abolish, incorporation of [ ³⁵ S]Met into newly synthesized proteins. (A and B) SL2 cells were transfected with GFP, REF1, REF2, DEK, SRm160, RNPS1, and Y14 dsRNAs. With the exception of cells depleted of NXF1, which were collected 4 d after transfection, depleted cells were collected 11 d after transfection. Cells were kept at 25°C (A) or subjected to a 1 h heat shock at 37°C (B). At this temperature, the heat-shock proteins (HSPs) are induced, whereas translation of nonheat shock proteins is inhibited. Cells were pulse labeled with [³⁵S]methionine for 1 h. Total lysates from equivalent numbers of cells were analyzed by SDS-PAGE followed by Coomassie blue stain and fluorography. Equal amounts of total protein were loaded per lane as judged by Coomassie staining. The efficacy of the depletions was tested by Western blot (not depicted). (C) SL2 cells were transfected with REF1 or REF2 dsRNAs. Aliquots of cells were collected 11 d posttransfection and analyzed by Western blotting with antibodies raised against the proteins indicated on the right. The steady-state expression level of REF1 was reduced, while the expression levels of DEK, NXF1, and UAP56 were not affected (compare lanes 1 and 2). In lanes 3–7, dilutions of the control sample were loaded to assess the efficiency of the depletion.

Figure 5.

Codepletion of EJC proteins reduces protein synthesis. (A) SL2 cells were transfected with GFP, NXF1, or REF1 dsRNAs or with a mixture of dsRNAs as indicated. Cell numbers were determined every two days up to 8 d after transfection. On day 4, cells were retransfected with the corresponding dsRNAs, with the exception of NXF1-depleted cells. Data are given as the n-fold increase in cell numbers relative to the amount of cells used for transfection on day 0 (where n represents a number on the y axis). (B) SL2 cells were transfected with GFP, NXF1, or REF1 dsRNAs or with a mixture of dsRNAs as indicated above the lanes. 8 d after transfection, cells were kept at 25°C (lanes 1–6) or subjected to a 1 h heat shock at 37°C (lanes 7–12) and pulse-labeled as described in Figure 6. Total cell extracts were analyzed by SDS-PAGE followed by Coomassie stain and fluorography. Equal amounts of total proteins were loaded per lane as revealed by Coomassie staining. (C) 8 d after transfection, cells from the experiment shown in B were either kept at 25°C (lanes 1–4) or subjected to a 1 h heat shock at 37°C (lanes 5–8). 10 μg of total RNA was analyzed by Northern blot using probes specific for rp49, hsp70 or hsp83 mRNAs (Herold et al., 2001). The bottom panel shows the corresponding rRNA stained with ethidium bromide. The asterisk indicates the position of the unspliced hsp83 precursor mRNA that accumulates at 37°C.

Figure 6.

Codepletion of EJC proteins leads to a partial nuclear accumulation of poly(A) ⁺ RNA. (A–H) SL2 cells were transfected with dsRNA corresponding to GFP, NXF1, and mixtures of dsRNAs. These mixtures consisted of dsRNAs corresponding to REF1 and RNPS1 or to REF1, RNPS1, SRm160, and Y14 as indicated. Cells depleted of NXF1 were fixed 5 d after transfection. Cells depleted of EJC proteins and control cells were fixed 8 d after transfection. Poly(A)⁺ RNA was detected by FISH with a Cy3-labeled oligo(dT) probe (A, C, E, G). The nuclear envelope was stained with Alexa 488-wheat germ agglutinin conjugates (B, D, F, H). The arrowheads point to cells in which a significant nuclear accumulation of poly(A)⁺ RNA was observed. Bar, 5 μm. (I–P) Representative examples of patterns of poly(A)⁺ RNA distribution in SL2 cells shown in A. Inhibition of mRNA export is complete in ∼94% of cells depleted of NXF1 (K), but partial with an equal distribution of the oligo(dT) signal between the nucleus and the cytoplasm in ∼20% of cells depleted of REF1 and RNPS1 (M). Depletion of all EJC proteins resulted in a stronger inhibition of mRNA export but a cytoplasmic signal was still detected (O). This phenotype is observed in ∼40% of the cell population. Bars, 2.5 μm.