Dbp5 does not displace Mex67 from exported pre-ribosomal subunits

of pre-ribosomal subunits, the question is how it contributes to this process. During mRNA export, Dbp5 is localized to the cytoplasmic fibrils and displaces bound export factors from the emerging mRNPs to provide directionality of the transport process (Lund and Guthrie, 2005; Tran et al., 2007). Thus, Dbp5 might also remodel the exported ribosomal particles and dissociate bound transport factors upon arrival in the cytoplasm. An obvious candidate to validate this hypothesis is Mex67, as its Dbp5-mediated displacement from exported mRNAs was already shown (Lund and Guthrie, 2005). Furthermore, Mex67 is also required for the transport of both pre-ribosomal subunits (Faza et al., 2012; Yao et al., 2007), but its cytoplasmic recycling mechanism is currently unknown and could be performed by Dbp5 (Gerhardy et al., 2014). To assess this question, sucrose-density gradient centrifugation was performed to analyze the ribosomal association of Mex67 in the dbp5 mutant rat8-2.

Figure 27: Mex67 is associated with polysomes and not displaced from ribosomal particles by Dbp5.

(A+B) Sucrose-density gradient fractionation experiments are shown with wild type (WT) and rat8-2 cells upon 1 h shift to 37°C. After cell lysis, half of the lysate was treated with 0.25 mg/ml RNase A and the untreated (A) and treated (B) lysates were loaded onto 7-47 % sucrose gradients and ultra-centrifuged. The gradients were fractionated while measuring the absorbance at 254 nm (A254nm) resulting in the profiles shown in the upper panels. The lower panels reveal the Western blot analysis of the corresponding proteins upon TCA precipitation. Mex67 and Rps3 were detected with specific antibodies. CBP80-GFP under control of a GAL-promoter was expressed by galactose induction and the tagged protein was detected with an anti-GFP antibody. The ratio of the proteins in the light (non-ribosomal+40S+60S) and the heavy (80S+polysomal) fractions compared to the total protein levels are indicated.

In principal, the translation rate was reduced in the dbp5 mutant rat8-2 (Figure 27A).

The reduction is characterized by a flattened polysome profile and an increased 80S peak along with the shift of the ribosomal protein Rps3 from the polysomes- to the

80S ribosomes-containing fractions (Figure 27A). Most likely, this reduction is caused by the mRNA export defects of rat8-2 and the decreased amount of translated mRNAs in the cytoplasm. This effect is also reflected by the shift of the cap-binding protein Cbp80, which is bound to the mRNA during the first rounds of translation (Garre et al., 2012), from the polysomal to the lighter fractions (Figure 27A).

Mex67 was part of the free 40S and 60S subunits-containing fractions of wild type and rat8-2 cells as expected for an export factor (Figure 27A). Furthermore, Mex67 was visible in the 80S ribosome-associated fractions of both strains. However, the 80S peak can also contain 90S particles so that the association with mature 80S ribosomes or 90S precursors is indistinguishable. Unexpectedly, Mex67 was also detectable in the polysomal fractions of wild type cells and its amount was even increased in rat8-2 (Figure 27A) indicating that Mex67 is either bound to the translated mRNAs or to the translating ribosomes. To distinguish between both possibilities, the lysates were treated with RNase A prior to loading of the gradients.

RNase A degrades the single-stranded mRNAs and thus, causes the disruption of the polysomes and formation of all translating ribosomes in one single 80S peak (see profiles of Figure 27B). Upon RNase A treatment, the mRNA-bound Cbp80 completely shifted to the non-ribosomal fractions confirming the successful mRNA degradation (Figure 27B). Nevertheless, Mex67 was still associated with the 80S ribosome-containing fractions in wild type and in rat8-2 cells (Figure 27B) showing that Mex67 is bound to the translating ribosomes even in wild type cells. Moreover, the amount of ribosome-associated Mex67 was not altered in rat8-2 compared to wild type (Figure 27B) and the same tendency was seen in initial experiments with rat8-7 (data not shown). Firstly, these results show that the enhanced amount of Mex67 in the polysomes of untreated rat8-2 cells results from the increased binding of Mex67 molecules to the translated mRNAs (Figure 27A). This increased association is most probably caused by the missing Mex67 release from the exported mRNAs in the dbp5 mutant (Lund and Guthrie, 2005). In addition, these data suggest that the ribosomal binding of Mex67, in contrast to its mRNA association is not influenced by Dbp5 and thus, Mex67 cannot be displaced by Dbp5 from the pre-ribosomal subunits.

To further support this idea, the binding of Mex67 to ribosomal proteins was analyzed in dbp5 mutants by co-immunoprecipitation experiments.

Figure 28: The ribosomal association of Mex67 is unchanged in the dbp5 mutants.

(A) Western blot analysis of an Rpl11b-GFP immunoprecipitation in wild type (WT) and rat8-7 cells reveals the same amount of co-precipitated Mex67 and Rps3 upon 1 h shift to 16°C. (B) Western blot analysis shows the same levels of co-precipitated ribosomal proteins Rps3 and Rpl35 in the Mex67-GFP pull-downs of wild type and rat8-2 cells. The cells were shifted for 1 h to 37°C. (A+B) All samples were treated with RNase A. Wild type cells without tagged proteins and detection of Aco1 (Aconitase) and Hem15 (Ferrochelatase), respectively served as non-binding controls.

As seen in Figure 28A, Mex67 was equally detectable in the immunoprecipitations of the large ribosomal protein Rpl11b-GFP in wild type and rat8-7 cells. Vice versa, Mex67-GFP co-precipitated the same amounts of the small ribosomal protein Rps3 and the large ribosomal protein Rpl35 in wild type and rat8-2 cells (Figure 28B).

Indeed, these data confirm that the association of Mex67 with ribosomal particles is not altered in the mutants of DBP5.

In summary, Dbp5 seems to displace Mex67 only from exported mRNAs, but not from ribosomal particles revealing differences in the transport mechanism of both large ribonucleoprotein particles.