Identification of novel ribonucleo-protein complexes from the brain-specific snoRNA MBII-52

Abstract

Small nucleolar RNAs (snoRNAs) guide nucleotide modifications within ribosomal RNAs or spliceosomal RNAs by base-pairing to complementary regions within their RNA targets. The brain-specific snoRNA MBII-52 lacks such a complementarity to rRNAs or snRNAs, but instead has been reported to target the serotonin receptor 2C pre-mRNA, thereby regulating pre-mRNA editing and/or alternative splicing. To understand how the MBII-52 snoRNA might be involved in these regulatory processes, we isolated the MBII-52 snoRNP from total mouse brain by an antisense RNA affinity purification approach. Surprisingly, by mass spectrometry we identified 17 novel candidates for MBII-52 snoRNA binding proteins, which previously had not been reported to be associated with canonical snoRNAs. Among these, Nucleolin and ELAVL1 proteins were confirmed to independently and directly interact with the MBII-52 snoRNA by coimmunoprecipitation. Our findings suggest that the MBII-52 snoRNA assembles into novel RNA-protein complexes, distinct from canonical snoRNPs.

FIGURE 1.

Antisense-RNA affinity purification of MBII-52. (A) Preparation of MBII-52 snoRNP from mouse brain. Solubility of MBII-52 upon lysis of nuclei (left) and immunoprecipitation of Fibrillarin from the lysate containing 300 mM NaCl (right). (B) An antisense RNA probe designed for affinity purification. (C, left) MBII-52 and Fibrillarin recovered from large-scale affinity purification. The quantity of MBII-52 was estimated from a standard curve using in vitro transcribed MBII-52 (not shown). Other abundant RNA species, i.e., U3 snoRNA and U6 snRNA, were not included in the eluate. (C, right) Proteins isolated from affinity-purified MBII-52 snoRNP. Different quantity of MBII-52 probe, indicated as + and ++, recovered ∼1 and 1.2 pmol of MBII-52 snoRNA, respectively. See Table 1 to find the corresponding protein of each band. NB, Northern blotting; WB, Western blotting.

FIGURE 2.

Candidate proteins closely related to MBII-52 RNP. (A) Distribution of candidate proteins in a glycerol gradient of brain nuclear extract. The signal peak of Nucleolin and ELAVL1, as well as Fibrillarin, appeared in lighter fractions than that of MBII-52 Northern blotting, while Matrin-3 and SNRPA appeared in heavier fractions. (B) MBII-52 copurified with an antibody against each candidate from fractions 3–8. Notably, MBII-52 was copurified in association with Fibrillarin from fractions 5 and higher. U6 signal was shown in the right for background control. (C) Immunoprecipitation of Nucleolin and ELAVL1 from individual fractions. Both proteins did not coimmunoprecipitate with each other. NB, Northern blotting; WB, Western blotting.

FIGURE 3.

Distribution of MBII-52 in nuclei. (A) Microscopic images (differential interference contrast [DIC]) during nucleoplasmic/nucleolar fractionation by sonication. Nuclei obtained from Neuro-2A cells were broken by five rounds of a 10-sec sonication, each. The RNA-enriched particles visualized by SYBR Green II staining are shown in the inset. Bars = 10 μm. (B) Monitoring the amount of nucleoplasmic/nucleolar components in collected pellets. MBII-52 exhibited a significant decline in its nucleoplasmic/nucleolar distribution close to nucleoplasmic U6 snRNA and c-fos protein. (C) Signal ratio of MBII-52/U3/U6 in supernatant to pellet. After a 30-sec sonication, fractions of supernatant and pellet, i.e., nucleoplasmic and nucleolar fraction, respectively, were purified and analyzed by Northern blotting. MBII-52 was enriched in nucleoplasmic fraction.

FIGURE 4.

A hypothetical model for MBII-52-protein assembly and function. A small portion of MBII-52 snoRNA may involve ELAVL1 in its complex and exert the function in nucleoplasm, while most MBII-52 assemble canonical C/D core proteins and localize to the nucleolus. Nucleolin may interact with the novel MBII-52 RNP and/or the canonical MBII-52 snoRNP to escort them for transit between nucleoplasm and nucleolus.