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. 1999 Feb 15;13(4):437-48.
doi: 10.1101/gad.13.4.437.

unr, a cellular cytoplasmic RNA-binding protein with five cold-shock domains, is required for internal initiation of translation of human rhinovirus RNA

S L Hunt  1 , J J Hsuan, N Totty, R J Jackson
Affiliations

unr, a cellular cytoplasmic RNA-binding protein with five cold-shock domains, is required for internal initiation of translation of human rhinovirus RNA

S L Hunt et al. Genes Dev. .

Abstract

Initiation of translation of the animal picornavirus RNAs occurs via a mechanism of direct ribosome entry, which requires a segment of the 5' UTR of the RNA, known as the internal ribosome entry site (IRES). In addition, translation of the enterovirus and rhinovirus (HRV) subgroups requires cellular trans-acting factors that are absent from, or limiting in rabbit reticulocytes, but are more abundant in HeLa cell extracts. It has been shown previously that HeLa cells contain two separable activities, each of which independently stimulates HRV IRES-dependent translation when used to supplement reticulocyte lysate; one of these activities was identified as polypyrimidine tract-binding protein (PTB). Here, the purification of the second activity is achieved by use of an RNA-affinity column based on the HRV 5' UTR. It comprises two components: a 38-kD protein (p38), which is a novel member of the GH-WD repeat protein family and has no intrinsic RNA-binding activity; and a 96- to 97-kD protein doublet, which was identified as unr, an RNA-binding protein with five cold-shock domains. Coimmunoprecipitation with antibodies against either protein shows that the two proteins interact with each other, and thus p38 is named unrip (unr-interacting protein). Recombinant unr acts synergistically with recombinant PTB to stimulate translation dependent on the rhinovirus IRES. In contrast, unr did not significantly augment the PTB-dependent stimulation of poliovirus IRES activity.

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Figures

Figure 1
Figure 1
HRV-2 5′ UTR RNA-affinity purification of HeLa cell B-type activity. HeLa cell B-type activity was partially purified by ion-exchange chromatography (see Materials and Methods) and then applied to an HRV 5′ UTR affinity column. Fractions 1–8 are flowthrough fractions, which were recycled over the matrix once and recollected as flowthrough fractions 9–16. Fractions 17–22 represent the 200 mm KCl wash, and fractions 23–41, the 200–1000 mm KCl gradient. (A) Silver-stained SDS/12.5% polyacrylamide gel of the column load (L) and fractions 1–41. (M) Protein molecular weight markers, of sizes indicated in kD in the left hand margin. (B) UV cross-linking of the column load (L) and fractions 1–40 to 32P-labeled HRV 5′ UTR RNA. A fluorogram of the 32P-labeled proteins, analyzed by SDS/15% polyacrylamide gel electrophoresis, is shown. UV cross-linking assays were carried out as described previously (Hunt and Jackson 1999) with 0.1 mg/ml heparin as a nonspecific competitor and a probe transcribed from pJHRV10-605, which consists of the complete HRV 5′ UTR (except for the first 9 nucleotides) plus the initiation codon, but without coding sequences. The reactions were carried out in the presence of 1 mg/ml BSA; in the absence of BSA (or 1 μl of reticulocyte lysate) the p97 signal was seen in precisely the same fractions but was weaker, presumably because of the low protein concentration. (C) Translation assays of the uncapped dicistronic XLJHRV10-611 mRNA in reticulocyte lysate, supplemented with 17% (vol/vol) column load (L) or fractions 22–35, which had been concentrated 10-fold by use of Microcon-10 microconcentrator units. (B) Negative control reaction supplemented with 17% (vol/vol) H500 buffer. An autoradiograph of the translation products analyzed by SDS/20% polyacrylamide gel electrophoresis is shown. The upstream cistron translation product (cyclin) and downstream cistron translation product (NS′) are indicated. (D) Purified B-type activity acts synergistically with recombinant PTB to stimulate HRV IRES-dependent translation. Uncapped dicistronic XLJHRV10-611 mRNA was translated in reticulocyte lysate in the presence of either 10 μg/ml recombinant GST–PTB (P), 16% (vol/vol) fraction 27 of the RNA affinity column from Fig. 1, prior to it being concentrated (B), or a combination of both (B+P). (C) Negative control reaction with no added factors. The yield of radiolabeled NS′ in each assay was determined by quantitative densitometry and is given below each lane, expressed relative to the yield in the buffer control assay, which was assigned a value of 1.0.
Figure 2
Figure 2
The deduced amino acid sequence of p38. Putative GH-WD motifs are underlined, and the conserved residues of the Asp-His-Ser/Thr structural triad identified by Sondek et al. (1996) in Gβ are shown in bold. The complete nucleotide sequence of the longest p38 cDNA is deposited in the EMBL/GenBank database (accession no. AJ 010025) and includes a putatively complete 3′ UTR of 495 nucleotides and a 147-nucleotide 5′ UTR.
Figure 3
Figure 3
A schematic representation of unr. Cold-shock domains are represented by black boxes, and the number of the first and last amino acid residues of each is indicated (numbering corresponds to the isoform lacking exon 5). The optionally spliced exon 5-encoded sequences are shown as a hatched box. The sequences of the conserved core of the five unr cold-shock domains which, by analogy with the cold-shock domain protein CspB (Schindelin et al. 1993; Schnuchel et al. 1993), are proposed to form the RNA-binding surface, are aligned below. Those residues that occur in four or more of the sequences are in bold.
Figure 4
Figure 4
Recombinant unr acts synergistically with PTB to stimulate HRV IRES-dependent translation. (A) Uncapped dicistronic XLJHRV10-611 mRNA was translated in reticulocyte lysate supplemented with recombinant His–unr, either lacking, or containing, the exon 5-encoded sequences (−exon5 and +exon5, respectively), at concentrations of 10, 5, 2.5, or 1.3 μg/ml, either separately, or in the presence of 10 μg/ml recombinant His–PTB. Control reactions without the addition of factors (C), or with the addition of 10 μg/ml His–PTB only (P), were also carried out. An autoradiograph of the translation products, analyzed by SDS/20% polyacrylamide gel electrophoresis, is shown. (B) The effect of recombinant unr, p38, and PTB on translation of HRV-2 genomic RNA. The truncated genomic HRV-2 transcript, HRV-2/NdeI, was translated in reticulocyte lysate supplemented with either 5 μg/ml recombinant His–unr (−exon 5), 20 μg/ml recombinant His–p38, or 10 μg/ml recombinant His–PTB, each separately, or in pairwise combinations, or all three together, as indicated above each lane. A negative control reaction was carried out without the addition of factors (C), and a positive control was supplemented with 20% (vol/vol) HeLa cell HS S100 extract (H). The yield of radiolabeled NS′ in each assay of A and the combined yield of P1-2A and P1 (plus any material of size intermediate between P1 and P1-2A) in B was determined by quantitative densitometry and is given below each lane expressed relative to the yield in the buffer control assay, which was assigned a value of 1.0.
Figure 5
Figure 5
The effect of HeLa cell B-type activity on poliovirus type 1 (Mahoney) IRES-dependent translation. (A) Uncapped dicistronic XLJHRV10-611 and XLPV1-747 mRNAs were translated in reticulocyte lysate supplemented with either 20% (vol/vol) partially purified HeLa cell B-type activity, fractionated from HeLa cell cytoplasmic HS S100 extract on heparin–Sepharose and then DEAE–Sepharose (B), 10 μg/ml recombinant His–PTB (P), or a combination of both (B+P). Negative control reactions were carried out with no addition of factors (C) and positive control reactions with the addition of 20% (vol/vol) HeLa cell HS S100 extract (H). An autoradiograph of the translation products, analyzed by SDS/20% polyacrylamide gel electrophoresis, is shown. The difference in size of the IRES-dependent cistron products is because NS′ in XLJHRV10-611 is a slightly truncated form of the NS-coding sequence fused directly to the viral initiation codon (Borman and Jackson 1992; Hunt and Jackson 1999), whereas the IRES-dependent cistron in XLPV1-747 is the full-length NS-coding sequence fused to the viral initiation site via a short linker (Hunt and Jackson 1999). (B) Poliovirion RNA (at 10 μg/ml) was translated in reticulocyte lysate in an assay supplemented with 10% (vol/vol) fraction 26 from the RNA-affinity column shown in Fig. 1 (after having been concentrated 10-fold). (C) Negative control reaction with no additional factors. The positions of the main polyprotein processing products (P1, P3, VP0, 2C, VP1, and VP3) are indicated at the right, as are the aberrant products (R and S) which arise from initiation events in the P3-coding region (Dorner et al. 1984). (C) Translation assays were carried out as in A, except that the mRNA was T7-1/NdeI. The translation product, P1′, is the P1 capsid precursor portion of the poliovirus polyprotein, slightly truncated at the carboxy-terminal end. The yield of radiolabeled NS′ or NS in each assay of A, and the yield of P1′ in each assay of C was determined by quantitative densitometry and is given below each lane, expressed relative to the yield in the corresponding buffer control assay, which was assigned a value of 1.0.
Figure 6
Figure 6
Comparison of the response of the HRV and poliovirus IRES elements to recombinant unr, PTB, and PCBP-2. Translation assays were carried out with either (A) uncapped XLJHRV10-611, (B) uncapped XLPV1-746, or (C) capped XLJHRV10-611 dicistronic mRNAs in reticulocyte lysate supplemented with 5 μg/ml His–unr (−exon 5), 10 μg/ml His–PTB, 20 μg/ml His–PCPB-2, and 20 μg/ml recombinant His–p38; each separately, or in various combinations as indicated above each lane. Control reactions were carried out either without the addition of factors (C) or supplemented with 20% (vol/vol) HeLa cell HS S100 extract (H). An autoradiograph of the translation products, analyzed by SDS/20% polyacrylamide gel electrophoresis, is shown. The yield of radiolabeled NS′ in A and C, or NS in B, was determined by quantitative densitometry and is given below each lane, expressed relative to the yield in the buffer control assay, which was assigned a value of 1.0.
Figure 7
Figure 7
unr and p38 coimmunoprecipitate. (A) Aliquots (5 μl) of HeLa HS S100 extract were separated by SDS/15% polyacrylamide gel electrophoresis, immunoblotted, and probed separately with either rabbit anti-p38 or rabbit anti-unr antibody (as indicated below each blot). (B) Antibodies covalently linked to protein A–Sepharose were used to pull out the specific antigens from HeLa cell HS S100 extract with either rabbit anti-p38, rabbit anti-unr antiserum, or the corresponding pre-immune serum, as indicated above each lane. The absorbed proteins were eluted with sample buffer, separated by SDS/15% polyacrylamide gel electrophoresis, immunoblotted, and the blot probed with a mixture of rabbit anti-p38 and rabbit anti-unr antibodies (as indicated below the blot). Detection was carried out with alkaline phosphatase-conjugated anti-rabbit secondary antibody. The smear of alkaline phosphatase activity across the upper half of the blot is caused by binding of anti-rabbit secondary antibody to primary antibody heavy chain, a small portion of which unavoidably leaches from the protein A–Sepharose matrix.
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