Molecular identification of p50 and p100. (A) Coomassie blue staining of purified p50 (lane 1) or p100 (lane 3) and their respective RNA–polypeptide complexes (lanes 2,4) detected by UV cross-linking and autoradiography. (B,C) Identification of YB-1 and nucleolin by the nanoelectrospray tandem mass spectra of doubly charged peptides extracted from the 50- and 100-kD bands, respectively. Sequence tags were assembled from a series of fragment ions and used to search for a matching pattern. Fragment masses calculated for the retrieved YB-1 GAEAANVTGPGGVPVQGSK or the retrieved nucleolin GYAFI/LEFASFEDAK sequences were compared with the complete fragmentation spectrum to confirm the match. (D) Schematic representation of nucleolin and YB-1. (Hatched boxes) RNA-binding domain; (dark grey box) RGG domain; (black boxes) RNP; (stippled boxes) cold shock domain.

In vitro regulation of IL-2 mRNA stability. (A) Stability of IL-2 mRNA in nonstimulated and stimulated extracts. ³²P-labeled, capped, and polyadenylated IL-2 and GAPDH RNAs were incubated with S100s from unstimulated or stimulated Jurkat cells at 37°C for various times. RNAs were isolated, resolved on a denaturing gel, and visualized by autoradiography. The amount of IL-2 RNAs was quantitated by phosphorimaging, normalized to the amount of GAPDH RNA, and plotted semilogarithmicly using a linear regression program. The values shown are averages of three independent experiments. (B) Stability of IL-2 RNA lacking nucleotides 550–680, including several AREs, in unstimulated or stimulated S100s was determined and quantitated as described as above. (○) Unstimulated; (●) stimulated. (C,D) In vitro IL-2 mRNA stabilization by JNK. S100 of unstimulated Jurkat cells was preincubated with recombinant activated JNK2, p38α, or ERK2, at 30°C for 20 min in the presence of ATP. RNA decay assays were performed as described above using wild-type IL-2 RNA (C) or IL-2 RNA containing linker scanning mutations (M1 or M4) within the 5′ UTR (D). (E,F) Quantitation of IL-2 RNA decay from three independent experiments identical to those shown in C and D, respectively. (E) (○) GST; (●) JNK + JNKK; (▴) p38 + MEK6; (█) ERK + MEK1. (F) (○) M1 + GST; (●) M1 + JNK + JNKK; (▴) M4 + GST; (█) M4 + JNK + JNKK.

Nucleolin and YB-1 are involved in IL-2 mRNA stabilization in vivo. (A) Jurkat cells were transfected with an IL-2 reporter, a control plasmid expressing luciferase (LUC) mRNA, and either MEKK1 or an empty expression vector along with constructs expressing YB-1 or nucleolin anti-sense RNAs. After 36 hr, actinomycin D (ActD) was added, total RNA was isolated at the indicated times, and the amount of IL-2 mRNA was analyzed by competitive RT–PCR. IL-2 mimic is a PCR product derived from a DNA template added to each reaction to control for amplification efficiency. IL-2 target is derived from transfected IL-2 mRNA. LUC mimic and target were also amplified. Each mimic and target were amplified with the same primer sets. (B) The amounts of nucleolin and YB-1 in Jurkat cells stably transfected with an empty vector (control) or antisense nucleolin RNA (N9). (C) Stability of endogenous IL-2 mRNA in control or N9 cells after stimulation with TPA + A23187. Total RNA was isolated at the indicated times after addition of ActD and the levels of IL-2 and β-actin mRNAs was determined by RNase protection. (D,E) Quantitation of exogenous and endogenous IL-2 mRNA decay in experiments as those shown in A and C, respectively. The quantitated IL-2 signals were normalized to the LUC and β-actin signals and plotted as described above. Each point is the average of three independent experiments. (D) (○) Vector; (●) MEKK1; (▴) MEKK1 + YB1 − AS; (█) MEKK1 + nucleolin-AS. (E) (●) Control; (○) N9.

Identification of JRE-binding proteins and critical sequences. (A) Schematic representation of wild-type and mutant IL-2 transcripts. (Open box) Coding region; (thin line) 5′ and 3′ UTRs; (solid boxes) AUUUA motifs. The signaling pathways involved in regulation of IL-2 mRNA stability are shown with their cis element targets. (B) UV cross-linking assay. Cytoplasmic extracts of Jurkat cells either unstimulated or stimulated with TPA (T), A23187 (A), or both (T/A) were incubated with ³²P-labeled IL-2(1–58) RNA. Following UV cross-linking, RNA–protein complexes were analyzed by denaturing SDS-PAGE and are indicated by arrows. (C) Specificity of RNA–protein complex formation. IL-2(1–58) RNA probe was coincubated with extracts of unstimulated Jurkat cells and the indicated molar excess of either homologous or heterologous RNA competitors (nonspecific 90-nucleotide transcript produced from the polylinker of pBluescript). (D) Sequences of the 5′ UTRs of wild-type and mutant IL-2 RNAs and their half-lives. CAT reporter genes flanked by IL-2 5′ and 3′ UTRs were cotransfected with MEKK1 or an empty expression vector into Jurkat cells and their t_1/2s were determined. The average t_1/2 (on the basis of two separate transfection experiments done in duplicate) of each RNA in the absence or presence of MEKK1 is indicated at right.

Binding of YB-1 and nucleolin to IL-2 RNA. (A) Jurkat cytoplasmic extracts were incubated with ³²P-labeled IL-2(1–58) RNA, UV cross-linked, and immunoprecipitated with control antibodies [mouse IgG (lane 2) or rabbit IgG (not shown)], a monoclonal antibody against nucleolin (lane 3), or polyclonal antibodies against YB-1 (lane 4). The input (25%, lane 1) and the precipitates were fractionated by SDS-PAGE, transferred to a membrane, and autoradiographed. These membranes were then probed with anti-nucleolin (lane 5) or anti-YB-1 (lane 6) antibodies and visualized by ECL. (B) UV cross-linking of wild-type and mutant IL-2(1–58) RNAs incubated with Jurkat extracts. (C) Binding of recombinant nucleolin or YB-1 to wild-type and mutant IL-2(1–58) RNAs. Each probe was incubated with GST–nucleolin(285–709) (100 ng) or GST–YB-1 (25 ng) and analyzed by UV cross-linking. (D) Specific binding of GST–nucleolin(285–709) or GST–YB-1 to IL-2(1–58) RNA. Wild-type ³²P-IL-2(1–58) RNA was incubated with nucleolin or YB-1 in the presence of molar excess of unlabeled wild-type or M2 IL-2(1–58) RNAs and analyzed by UV cross-linking. (E) In vivo association of IL-2 mRNA with nucleolin and YB-1. The proteins were immunoprecipitated from either unstimulated (NS) or stimulated (T/A) Jurkat cells. Portions of the precipitates were analyzed by immunoblotting (IB) with either anti-YB-1 or anti-nucleolin (top). RNA was extracted from the immune complexes (ppt) and analyzed by RT–PCR and Southern blotting. RNA was also extracted from supernatants (sup) and similarly analyzed. (F) In vivo association of nucleolin and YB-1 with wild-type or IL-2 mRNA mutants. Jurkat cells were transfected with vectors expressing either wild-type IL-2 mRNA or a mutant in which the IL-2 5′ UTR was removed (Δ5′-UTR) or CAT reporters, containing wild-type or mutant 5′ UTRs, described in Fig. 1D. After 48 hr, cells were stimulated, lysed, and immunoprecipitated with anti-nucleolin or anti-YB-1. The presence of reporter-derived transcripts in the immune complexes was analyzed by RT–PCR. (Top) Expression of each transcript in transfected cells; (middle, bottom) precipitated RNAs.

Nucleolin and YB-1 are required for JNK-induced IL-2 RNA stabilization in vitro. (A) Immunoblot analysis with anti-nucleolin (lanes 1–5), anti-YB-1 (lanes 6–10), or anti-La (lanes 11,12) of Jurkat S100 fraction that was untreated (lanes 1,4,6,9,11) or passed (three times) through protein A-agarose beads containing anti-nucleolin (lanes 2,7), anti-YB-1 (lanes 3,8), or anti-La (lanes 5,10,12). (B) The immunodepleted supernatants were preincubated with activated JNK2 (JNKK+JNK), purified nucleolin or YB-1, or activated JNK2 plus nucleolin or YB-1 (as indicated) and used to examine the decay of wild-type IL-2 RNA. (C,D) Quantitation of IL-2 RNA decay in three independent experiments identical to those shown in B. (C) (○) anti-La + GST; (●) anti-La + JNKK + JNK; (▴) anti-nucl. + JNKK + JNK; (▪) anti-YB1 + JNKK + JNK. (D) (○) anti-nucl. + nucl.; (●) anti-nucl. + nucl. + JNKK + JNK; (▴) anti-YB1 + YB1; (█) anti-YB1 + YB1 + JNKK + JNK.

Restoration of IL-2 mRNA stabilization in N9 cells by exogenous nucleolin. (A) N9 cells were cotransfected with the IL-2 reporter and expression vectors as indicated. The stability of IL-2 mRNA in the transfected cells was determined as described in Fig. 6A. (B) IL-2 mRNA stabilization in response to stimulation (T/A) of parental or N9 Jurkat cells transfected with either a nucleolin or an empty expression vector. (C,D) Quantitation of IL-2 mRNA decay in A and B. Each point is the average of two transfection experiments. Inset in D shows expression of HA-tagged nucleolin in transfected N9 cells. (C) (○) Vector; (●) nucleolin; (▴) MEKK; (█) MEKK + nucleolin. (D) (○) Jurkat + T/A; (▴) N9; (●) N9 + T/A; (█) N9 + nucleolin + T/A.