Chimeric human CstF-77/Drosophila Suppressor of forked proteins rescue suppressor of forked mutant lethality and mRNA 3' end processing in Drosophila

Abstract

The Suppressor of forked [Su(f)] protein is the Drosophila homologue of CstF-77, a subunit of human cleavage stimulation factor (CstF) that is required for the first step of the mRNA 3' end processing reaction in vitro. We have addressed directly the role of su(f) in the mRNA 3' end processing reaction in vivo. We show that su(f) is required for the cleavage of pre-mRNA during mRNA 3' end formation. Analysis of the functional complementation between Su(f) and CstF-77 shows that most of the Drosophila protein (85%) can be exchanged for the human protein to produce chimeric CstF-77/Su(f) proteins that rescue lethality and cleavage defect during mRNA 3' end formation in su(f) mutants. Interestingly, we show that a domain in human CstF-77 is limiting for the rescue and that this domain is not able to reproduce protein interactions with the CstF subunits of Drosophila. We also show that chimeric CstF-77/Su(f) proteins that rescue lethality of su(f) mutants cannot restore utilization of a regulated poly(A) site in Drosophila. Taken together, these results demonstrate that CstF-77 and Su(f) have the same function in mRNA 3' end formation in vivo, but that these two proteins are not interchangeable for regulation of poly(A) site utilization.

Fig 1.

Rescue of su(f) mutants with Su(f), CstF-77, or chimeric CstF-77/Su(f) proteins. (A) Comparison of Su(f) and CstF-77 proteins. Vertical lines indicate identical residues, colons represent similarities, and single dots represent less similar residues. Open boxes are tetratricopeptide-like repeats, the gray box indicates the nuclear localization signal, and the dashed box is the proline-rich domain. Arrows indicate the positions where Su(f) and CstF-77 are fused in the different chimeric proteins. (B) Rescue of su(f) mutants with Su(f), CstF-77, or chimeric CstF-77/Su(f) proteins. All of the transgenes were expressed by using the UAS/Gal4 system with the da-Gal4 driver. The rescue was assayed at 25°C for su(f)^R-9–18, su(f)^3DES, and su(f)^L26 and at 29°C for su(f)^ts67g. +, Viable adults; −, lethal; nd, not determined. The numbers indicate the number of transformant lines that rescue out of the number of transformant lines tested.

Fig 2.

Protein interactions within Drosophila CstF and between human CstF-77 and Drosophila CstF subunits. (A) Schematic representation of Drosophila CstF subunits and GST fusion proteins. For each GST fusion protein, the part of the Drosophila protein fused to GST is indicated. The internal domain of Su(f), containing the proline-rich (Pro) and hinge domains, that was exchanged in chimeric CstF-77/Su(f) proteins is indicated (I). TPR, tetratricopeptide repeats; NLS, nuclear localization signal; RBD, RNA binding domain. (B) GST pull-down assays. ³⁵S-labeled Su(f), CstF-77, chimeric CstF-77/Su(f), or Drosophila CstF-50 proteins were incubated with GST or GST fusion proteins. Incubations were in the presence of 40 μl of glutathione-Sepharose beads. Labeled proteins eluted from the beads, as well as 10% of the input-labeled proteins (input), were analyzed by electrophoresis and quantified with image quant. Amounts are indicated in percentages, relative to the input. Lane 1, input; lane 2, GST; lane 3, GST-Su(f)492–733; lane 4, GST-CstF-64; lane 5, GST-CstF-64H; lane 6, GST-CstF-50; lane 7, GST-CstF-50WD.

Fig 3.

Rescue of the cleavage defect in the su(f)^L26 mutant by chimeric CstF-77/Su(f) proteins. (A) Sequences of rp49 and sop poly(A) site regions. Potential poly(A) signals are in bold. Vertical arrows indicate poly(A) sites determined from cDNA sequences in databases. GU/U-rich sequences are underlined. Horizontal arrows indicate primers used for the PCR. (B) RT-PCR assays. The control pgk PCR fragment is generated with primers on each side of intron 2 of pgk. The size of the pgk PCR product we obtained (434 bp) was the expected size for amplification of pgk RNA after splicing of intron 2. rp49 (369 bp) and sop (316 bp) PCR fragments are obtained with primers indicated in A, only if no cleavage occurs at the poly(A) site. Lane 1, wild type; lane 2, su(f)^L26; lane 3, su(f)^L26;da-Gal4; lane 4, su(f)^L26;UAS-CstF-77-C/+;da-Gal4/+; lane 5, su(f)^L26;UAS-CstF-77-N/da-Gal4; lane 6, su(f)^L26;UAS-su(f)/da-Gal4; lane 7, su(f)^L26;UAS-CstF-77/da-Gal4.

Fig 4.

Utilization of the su(f) intronic poly(A) site in the su(f)^ts67g mutant is not restored with the chimeric CstF-77/Su(f) proteins. (A) Structure of the su(f) locus and RNAs. Open boxes are noncoding sequences and introns, black boxes are coding sequences. Vertical arrows indicate poly(A) sites. (B) RNA blots with poly(A)⁺ RNA from wild-type and su(f)^ts67g adult males raised at 25°C and shifted for 4 days to 29°C were hybridized with a su(f) RNA probe specific to the 1.3-kb RNA. This probe is complementary to 88 nt located in the part of intron 4 incorporated in the 1.3-kb RNA. The blots were reprobed with the rp49 clone as a loading control. Quantification was performed with National Institutes of Health image. The 1.3-kb/rp49 ratios are indicated relative to the value obtained for the wild type, set at 1. Lane 1, wild type; lane 2, su(f)^ts67g; lane 3, su(f)^ts67g;da-Gal4; lane 4, su(f)^ts67g;UAS-su(f)/da-Gal4; lane 5, su(f)^ts67g;UAS-CstF-77-N/da-Gal4; lane 6, su(f)^ts67g;UAS-CstF-77-C/+; da-Gal4/+.