Structural organization of mRNA complexes with major core mRNP protein YB-1

Abstract

YB-1 is a universal major protein of cytoplasmic mRNPs, a member of the family of multifunctional cold shock domain proteins (CSD proteins). Depending on its amount on mRNA, YB-1 stimulates or inhibits mRNA translation. In this study, we have analyzed complexes formed in vitro at various YB-1 to mRNA ratios, including those typical for polysomal (translatable) and free (untranslatable) mRNPs. We have shown that at mRNA saturation with YB-1, this protein alone is sufficient to form mRNPs with the protein/RNA ratio and the sedimentation coefficient typical for natural mRNPs. These complexes are dynamic structures in which the protein can easily migrate from one mRNA molecule to another. Biochemical studies combined with atomic force microscopy and electron microscopy showed that mRNA-YB-1 complexes with a low YB-1/mRNA ratio typical for polysomal mRNPs are incompact; there, YB-1 binds to mRNA as a monomer with its both RNA-binding domains. At a high YB-1/mRNA ratio typical for untranslatable mRNPs, mRNA-bound YB-1 forms multimeric protein complexes where YB-1 binds to mRNA predominantly with its N-terminal part. A multimeric YB-1 comprises about twenty monomeric subunits; its molecular mass is about 700 kDa, and it packs a 600-700 nt mRNA segment on its surface.

Figure 1

Sedimentation and density analysis of YB-1 complexes with α-globin mRNA. (A) Sedimentation distribution of YB-1 complexes with α-globin mRNA in sucrose gradient. YB-1 was mixed with ³²P-labeled α-globin mRNA at indicated molar ratios. The formed complexes were layered on 5–20% sucrose gradient and centrifuged in a SW-60 rotor at 45 000 r.p.m. for 4 h at 4°C. UV absorbance at 254 nm (solid line) and radioactivity (open circles) are shown. YB-1 distribution over gradient fractions revealed by SDS–gel electrophoresis is shown below each figure. (B) Distribution of YB-1 complexes with α-globin mRNA in CsCl density gradient. YB-1 complexes with ³²P-labeled α-globin mRNA formed at indicated molar ratios were fixed with 4% formaldehyde. Then the samples were centrifuged in CsCl gradient in a SW-55Ti rotor at 36 000 r.p.m. for 24 h at 4°C. Radioactivity (open circles) and CsCl density (filled triangles) are shown. YB-1 distribution over gradient fractions revealed by immunoblotting with antibodies to YB-1 is shown below each figure.

Figure 2

(A) Alteration in the multimeric state of YB-1 within its complexes with α-globin mRNA formed at different ratios of YB-1/mRNA. Complexes of YB-1 with ³²P-labeled α-globin mRNA were formed at indicated YB-1/mRNA ratios and fixed with 0.15% glutaraldehyde. RNA was destroyed by RNases A and T1 and by boiling with Mg²⁺ ions. The protein was analyzed using SDS–gel electrophoresis. Left, YB-1 without mRNA: lane 1, without fixation; lane 2, after fixation with glutaraldehyde, Coomassie stained. Middle and right, YB-1 from YB-1/mRNA complexes formed at indicated ratios of the components: lane 1, without addition of RNA; lanes 1–3, without fixation; lanes 4–9, after fixation with glutaraldehyde. Middle, Coomassie stained; right, radioautograph. (B) Analysis of domain YB-1 contacts with α-globin mRNA within mRNP complexes. The scheme presents the domain organization of YB-1 and distribution of methionine residues. Complexes of YB-1 and ³²P-labeled α-globin mRNA were formed at indicated molar ratios, exposed to UV and treated with RNases A and T1. YB-1 was cleaved with cyanogen bromide at methionine residues. Left, gel autoradiograph after SDS–gel electrophoresis of YB-1 fragments resulting from cleavage is shown. Right, the graph demonstrates changes in the relative amount of radioactive RNA cross-linked with fragment I and fragment II of YB-1.

Figure 3

Analysis of stability of YB-1 complexes with α-globin mRNA. Complexes of YB-1 with ³²P-labeled α-globin mRNA were formed at YB-1/mRNA molar ratios of 36:1. The sample was incubated for 15 min at 30°C, then unlabeled α-globin mRNA was added to a half of the sample to the final ratio of 9:1, and the preparations were incubated for additional 15 min at the same temperature. The preparations were layered onto 5–20% sucrose gradient and centrifuged in a SW-60 rotor at 45 000 r.p.m. for 4 h at 4°C. (A) Distribution of the complex formed by YB-1 with α-globin mRNA at a molar ratio of 36:1. (B) Distribution of the complex formed by YB-1 with α-globin mRNA at a molar ratio of 36:1 to which unlabeled α-globin mRNA was added to the YB-1/mRNA ratio of 9:1. UV absorbance (solid line) at 254 nm and radioactivity (open circles) profiles are shown. YB-1 distribution over gradient fractions revealed by SDS–gel electrophoresis is shown below the plots.

Figure 4

EM and AFM images of free multimeric and monomeric YB-1. (A) Electron micrograph of multimeric YB-1. The protein, fixed with 0.15% glutaraldehyde, was adsorbed onto a carbon film and shadowed with platinum-carbon. The same images of shadowed fixed YB-1 were obtained at its adsorbtion onto mica (not shown). (B) Electron micrograph of negatively stained fixed YB-1 deposited on mica. (C) AFM image of fixed YB-1 on APS-mica imaged in air. An aliquot of 5 μl of fixed YB-1 (0.01 μg/μl) was deposited onto the substrate, rinsed with water, dried and imaged. (D) AFM image of YB-1 in high salt solution on APS-mica substrate. YB-1 (0.05 μg/μl) was incubated in a high salt buffer (10 mM HEPES–KOH pH 7.6, 1 M LiCl) for 20 min at 30°C. Cross-sections made along the marked lines in (C) and (D) are shown under the respective AFM images. These cross-sections represent the height of the complexes and its variations. Triangles in the fields indicate the ends of the section lines.

Figure 5

AFM images of unsaturated YB-1–mRNP complexes in air. Unsaturated complexes of YB-1 with α-globin mRNA (A) and 2Luc mRNA (B) were fixed with glutaraldehyde, deposited on APS-mica and imaged as described in Materials and Methods. Left, typical field; right, magnified images of individual complexes and cross-sections made along the marked lines on the fields. Triangles in the fields indicate the ends of the section lines.

Figure 6

AFM images of saturated mRNP complexes. Fixed saturated complexes of YB-1 with α-globin mRNA (A), 2Luc mRNA (B) and TMV RNA (C) were deposited on APS-mica and imaged in air. Left, typical field; right, cross-sections made along the marked lines on the fields. Triangles in the fields indicate the ends of the section lines. Histograms illustrating the distribution of number of multimeric globules per complex are shown on the right.

Figure 7

Images of saturated complexes formed with 2Luc mRNA at high magnification. (A) AFM images of individual complexes in air. Right, cross-sections made along the marked lines on the corresponding images. (B) Electron micrograph of the negatively stained complexes.

Figure 8

Scheme showing involvement of YB-1 in the formation of polysomal and free cytoplasmic mRNPs. For details, see text.