|
Fig. 1.
Full length Xqua cDNA sequence. The 5′ and 3′ UTR sequences (in lower case) are 599 nt and 2819 nt long respectively, with an ORF of 1095 nt (in upper case). The conceptual translation of the ORF is presented below. The extended KH domain is contained in parentheses. The 24 nt alternatively spliced sequence has a bold line over it. The 3′ UTR contains eleven putative mRNA destabilizing sequences, AUUUA ( Shaw and Kamen, 1986; Wilson and Treisman, 1988), which are underlined. A polyadenylation signal sequence (AAUAAA) is indicated in bold upper case. Clones containing Xqua sequences were initially identified using a 1.1 kb fragment of the mouse qkI coding region to probe 106 clones from a stage 30 Xenopus head cDNA library ( Hemmati-Brivanlou et al., 1991). Hybridizations were performed in 40% formamide, 1 M NaCl, 1% SDS, 50 mM Tris-HCl (pH 7.6), 5×Denhardt's and 100 μg/ml herring sperm DNA, at 42°C. Filters were washed at moderate stringency, in 2×SSC/0.1% SDS at 55°C. Exonuclease III digestion (Erase-a-Base, Promega) was used to make nested deletions of overlapping Xqua cDNA clones and sequence was determined by chain-termination sequencing ( Sanger et al., 1977) of both strands of the Xqua cDNA.
|
|
Fig. 2.
Comparison of the Xenopus and mammalian quaking sequences. (A) Comparison of Xenopus, mouse and human 5 kb qk cDNA sequences, showing percentage nt identity for different regions. (B) Alignment of Xenopus, mouse and human quaking aa sequences. Identical residues are indicated by a dash (–) and the stop codon by an asterisk (*). The standard KH domain is boxed and the extended KH domain is included in parentheses. The region of similarity found in the GSG/STAR subfamily is highlighted in gray. The sequences that can be alternatively spliced in Xenopus and human are indicated by a bold overline. The human ORF was compiled from overlapping ESTs and spans a region equivalent to pXqua aa 103–318. (C) Alignment of the three blocks of the 3′ UTR sequence that are highly conserved between Xenopus, mouse and human. The Xenopus sequence is presented above with identical nt indicated by a dash (–), spaces in the sequence represent deletions or insertions and unknown sequence is indicated by (?). The accession numbers for human EST entries containing qk sequences are: T78719, R21227, R1228, R63053, R62996, R12087, R36880, R12116, R36870, R25054, R45393, R51027, H05147, H16837, Z28670, Z24967, T34625, R57586, R57984, R58351, Z33552, H47907, T82267, T83554, H41198, H41197, H41765, H41764, H49924, H50457, H47907, H47906, H66634, H87919, R51134, H05190, D59576, D62391. Sequences were multiply aligned using CLUSTAL V ( Higgins et al., 1992) and Assembly-Lign (IBI Inc).
|
|
Fig. 3.
Detection of quaking sequences in Xenopus embryonic mRNA. (A) Northern blot analysis of 10 μg poly(A)+RNA from Xenopus neurula, (stage 15), tailbud (stage 25) and tadpole (stage 35) embryos, probed with Xenopus quaking sequences detects two transcripts 5 kb and 5.5 kb in length. The probe cDNA fragment is derived from the coding region and spans the KH domain (nt 1–1718). (B) The same RNA blot probed with Xqua cDNA fragments derived from the 3′ UTR (nt 1718–2240 or nt 2450–3020), detects only the 5 kb mRNA. (C) Expression profile of the 5 kb Xqua transcript. Total RNA from five embryos at each stage was analyzed by RNase protection, using probe specific for the 5 kb Xqua transcript (nt 2046–2240), or probe for the control sequence XMax, which is expressed at constant levels throughout development ( Tonissen et al., 1994). Total RNA was isolated from Xenopus eggs and embryos as described by Melton and Cortese (1979)and purified further by precipitation with 4 M LiCl. Northern blot analysis was performed using random primed probes from three different Xqua cDNA fragments: (1) Xqua, NotI-EcoRI fragment, nt 1–1718; (2) Xqua, EcoRI fragment, nt 1718–2240; (3) Xqua, HindIII fragment, nt 2450–3020. Plasmid for generating Xqua 5 kb specific, antisense RNA probe was constructed by inserting a 520 bp EcoRI Xqua fragment (nt 1718–2240) into pBluescript SK (Stratagene). The template was linearized with PvuII and 32P-labeled probe was transcribed using T7 RNA polymerase. RNase protection assays were performed as described by Krieg and Melton (1987)with the Xqua probe and probe from the XMax-2 template, p4Z.MaxAH ( Tonissen and Krieg, 1994).
|
|
Fig. 4.
pXqua binds RNA in vitro. Binding of (A) pXqua357 and (B) pXqua365 to RNA in vitro. Synthetic mRNA encoding pXqua357 or pXqua365 was translated in vitro in reticulocyte lysates in the presence of [35S]methionine. An amount equal to 20% of the protein(s) used in each binding reaction is shown in the track marked translation. 35S-labeled proteins were bound to 30 μg of total Xenopus embryonic RNA-agarose (track labeled +RNA) in buffer containing 100 mM NaCl. As a control, an equal amount of protein was bound to agarose beads that were not coupled to RNA (track labeled −RNA). After extensive washing, protein retained on the RNA-agarose was analyzed by SDS-PAGE using a 12.5% gel and visualized by fluorography. The control CAT protein did not bind to the RNA-agarose. To generate labeled protein for RNA-binding studies, sequences encoding pXqua357 and pXqua365 were inserted into pT7TS vector (a modified version of pSP64T, Krieg and Melton, 1987). pT7TS-Xqua templates were linearized with BamHI and capped mRNA was produced using the Ambion Message Machine kit. Approx. 100 ng of synthetic mRNA encoding pXqua357, pXqua365 or pCAT (control sequence provided by BRL) was translated in vitro in the presence of [35S]methionine, using reticulocyte lysate (Ambion). To generate RNA-agarose, total Xenopus embryonic RNA at 1 mg/ml in water was biotinylated using photo-activated biotin-acetate (Vector Labs) according to the manufacturer's instructions. The RNA-biotin was then mixed with streptavidin-agarose resin (BRL) to a final concentration of 1–2 μg of RNA per 1 μl of agarose resin. RNA binding of in vitro translated proteins was assayed essentially as described by Swanson and Dreyfuss (1988)using approx. 105 dpm of labeled protein and 30 μl RNA-agarose resin. Proteins bound to the RNA-agarose were released by addition of SDS-protein loading buffer, boiled, and then resolved by SDS-PAGE and visualized by fluorography. In each experiment, control binding reactions were carried out with streptavidin-agarose (inactivated) that was not coupled to RNA.
|
