Identification of two RNA-binding proteins associated with human telomerase RNA - PubMed (original) (raw)

. 2000 Mar;11(3):999-1010.

doi: 10.1091/mbc.11.3.999.

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Free PMC article

Identification of two RNA-binding proteins associated with human telomerase RNA

S Le et al. Mol Biol Cell. 2000 Mar.

Free PMC article

Abstract

Telomerase plays a crucial role in telomere maintenance in vivo. To understand telomerase regulation, we have been characterizing components of the enzyme. To date several components of the mammalian telomerase holoenzyme have been identified: the essential RNA component (human telomerase RNA [hTR]), the catalytic subunit human telomerase reverse transcriptase (hTERT), and telomerase-associated protein 1. Here we describe the identification of two new proteins that interact with hTR: hStau and L22. Antisera against both proteins immunoprecipitated hTR, hTERT, and telomerase activity from cell extracts, suggesting that the proteins are associated with telomerase. Both proteins localized to the nucleolus and cytoplasm. Although these proteins are associated with telomerase, we found no evidence of their association with each other or with telomerase-associated protein 1. Both hStau and L22 are more abundant than TERT. This, together with their localization, suggests that they may be associated with other ribonucleoprotein complexes in cells. We propose that these two hTR-associated proteins may play a role in hTR processing, telomerase assembly, or localization in vivo.

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Figures

Figure 1

Figure 1

Structural alignment and sequence comparison of domains in hStau and other double-stranded RNA (dsRNA)-binding proteins. (A) Structure of hStau and other proteins that contain double-stranded RNA-binding domains. Full-length dsRNA-binding domains are indicated by gray boxes, and short domains are indicated by white boxes. The numbers under the domains in hStau and Staufen correspond to the sequences listed in B. (B) Sequence homology between double-stranded RNA-binding domains of human Staufen and Drosophila Staufen (STAU). Top and Middle, alignment of the full-length domains. Bottom, alignment of the short domains. Identical residues are shaded; * symbols indicate the residues that are highly conserved in most of the double-stranded RNA-binding domains containing proteins (St. Johnston et al., 1992) (note that not all sequences are shown). Sequences used in the alignment are as follows: hStau-1, aa 59–79; hStau-2, aa 100–172; hStau-3, aa 202–275; hStau-4, aa 452–472; STAU-1, aa 308–380; STAU-2, aa 490–559; STAU-3, aa 575–647; STAU-4, aa 708–782; and STAU-5, aa 948-1020.

Figure 2

Figure 2

hStau and L22 interact with hTR. (A) hStau antibody specificity. Western blot analysis on a 293 cell extract (lane 1) and hStau immunoprecipitates (lane 2) using anti-hStau antibody shows the 55-kDa hStau protein. The relative mobility of molecular weight markers (in kilodaltons) is indicated on the left. (B) L22 antibody specificity. Western blot analysis on 293 cell extracts (lane 1) and L22 immunoprecipitates (lanes 2–4) is shown. Lane 2, precipitation using preimmune serum (pre); lane 3, precipitation using anti-L22 serum; and lane 4, preincubation of L22 peptide (pep) with anti-L22 serum before immunoprecipitation. The relative mobility of molecular weight markers (in kilodaltons) is indicated on the left. The arrow indicates the L22 band (15 kDa). The IgG heavy and light chains (arrowheads) are also indicated. (C) RT-PCR analysis of RNAs in the supernatant (Sup; lanes 1–7) and pellet (lanes 8–14) fractions of hStau and L22 immunoprecipitation reactions. Lanes 1 and 8, hStau precipitation using preimmune serum; lanes 2 and 9, precipitation using anti-hStau antibody; lanes 3 and 10, precipitation using anti-GST antibody; lanes 4 and 11, precipitation using anti-chymotrypsin (-chym.) antibody; lanes 5 and 12, precipitation using L22 preimmune serum; lanes 6 and 13, precipitation using anti-L22 antibody; and lanes 7 and 14, precipitation using anti-L22 antibody preincubated with L22 peptide. The RNAs amplified in the fractions are indicated on the left (hTR, U2, U3, 7SL, and RNase P). (D) Quantitation of RNAs precipitated in C. The RNA precipitated is expressed as a fraction of the total signal intensity obtained in both the pellet and supernatant. RNAs assayed are indicated on the left, and antibodies used are indicated on the bottom. L22+pep, L22 antibodies preincubated with the L22 peptide before immunoprecipitation. a.u., arbitrary units.

Figure 3

Figure 3

Anti-Sm and anti-SF2 antibodies do not precipitate hTR. (A) Quantitation of hTR precipitated as described in Figure 2D. Antibodies used are indicated on the bottom. (B) Western blot analysis on 293 cell extracts (lane 2) and an Sm immunoprecipitate (lane 1). The arrow indicates Sm proteins (28–29 kDa) recognized by mAb Y12. IgG bands (arrowheads) are also indicated on the right. (C) Western blot analysis on 293 cell extracts (lane 2) and SF2 immunoprecipitates (lane 1). The arrow indicates the SF2 protein (33 kDa). IgG bands (arrowheads) are also indicated on the right.

Figure 4

Figure 4

hStau and L22 interact with telomerase and hTERT. (A) hStau and L22 associate with telomerase activity in 293 cells. Telomerase assays (modified TRAP, see MATERIALS AND METHODS) were performed on a 293 extract (assayed at 5 μg of protein; lane1) and immunoprecipitation pellets (lanes 2–13) from immunoprecipitations with various antibodies. The antibodies used are indicated at the top of the gel. Lanes 2 and 3, precipitation using hStau preimmune serum; lanes 4 and 5, precipitation using anti-hStau antibody; lanes 6 and 7, precipitation using anti-GST antibody; lanes 8 and 9, precipitation using L22 preimmune serum; lanes 10 and 11, precipitation using anti-L22 antibody; and lanes 12 and 13, precipitation using anti-L22 antibody preincubated with L22 peptides. Samples in lanes 3, 5, 7, 9, 11, and 13 were pretreated with RNase (+) before the telomerase reactions. (B) hStau and L22 interact with hTERT. Western blot analysis using anti-HA (top), anti-hStau (middle), or anti-L22 (bottom) antibody on cell lysates (lanes 1 and 2) or various immunoprecipitation pellets (lanes 3–8) is shown. Extracts used are a mock-transfected 293 extract (lanes 1, 3, and 6) and an hTERT-HA–transfected cell extract (lanes 2, 4, 5, 7, and 8). The antibodies used in immunoprecipitation reactions were the following: lanes 3 and 5, precipitation using anti-L22; lane 4, precipitation using L22 preimmune serum; lanes 6 and 8, precipitation using anti-hStau; and lane 7, precipitation using hStau preimmune serum. The relative mobility of molecular weight markers (in kilodaltons) is indicated on the left. The identity of various bands is indicated by arrows.

Figure 5

Figure 5

Subcellular localization of the hStau and the L22 protein. (A) Green fluorescence of GFP-hStau and nucleolin immunofluorescence of 293 cells expressing the GFP-hStau fusion. Cells were stained with DAPI (blue, top), green fluorescence (green, second from top), and anti-nucleolin (red, second from bottom), and images were merged (bottom). (B) Green fluorescence of GFP-hStau and L22 immunofluorescence of 293 cells expressing the GFP-hStau fusion. Cells were stained with DAPI (blue, top) and L22 antiserum (red, bottom) and processed for a green fluorescence image (green, middle).

Figure 6

Figure 6

Interaction between telomerase-associated proteins. (A) hStau and L22 do not interact with each other. A 293 cell lysate was immunoprecipitated with hStau preimmune serum (lane 2), hStau antibody (lane 3), L22 preimmune serum (lane 4), or L22 antibody (lane 5). The pellet fractions were analyzed by Western hybridization probed with either hStau antibody (top) or L22 antibody (bottom). (B) hStau and myc-TEP1 do not interact. 293 cells were either mock transfected (lanes 1, 3, and 4) or transfected with the myc-TEP1 construct (lanes 2, 5, and 6). Cell lysates were either run directly on a Western (lanes 1 and 2) or immunoprecipitated with either hStau preimmune serum (lanes 3 and 5) or hStau antibody (lanes 4 and 6). The Western was probed with anti-myc antibody (top) or hStau antibody (bottom). (C) L22 and myc-TEP1 do not interact. 293 cells were either mock transfected (lanes 1 and 3) or transfected with the myc-TEP1 construct (lanes 2, 4, and 5), and cell lysates were either run directly on a Western (lanes 1 and 2) or immunoprecipitated with either hStau preimmune serum (lane 4) or L22 antibody (lanes 3 and 5). The Western was probed with anti-myc antibody (top) or L22 antibody (bottom). The relative mobility of molecular weight markers (in kilodaltons) is indicated on the left in A–C.

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