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. 2014 Jul;20(7):1090-102.
doi: 10.1261/rna.045005.114. Epub 2014 May 23.

Identification of the RNA Recognition Element of the RBPMS Family of RNA-binding Proteins and Their Transcriptome-Wide mRNA Targets

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Free PMC article

Identification of the RNA Recognition Element of the RBPMS Family of RNA-binding Proteins and Their Transcriptome-Wide mRNA Targets

Thalia A Farazi et al. RNA. .
Free PMC article

Abstract

Recent studies implicated the RNA-binding protein with multiple splicing (RBPMS) family of proteins in oocyte, retinal ganglion cell, heart, and gastrointestinal smooth muscle development. These RNA-binding proteins contain a single RNA recognition motif (RRM), and their targets and molecular function have not yet been identified. We defined transcriptome-wide RNA targets using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) in HEK293 cells, revealing exonic mature and intronic pre-mRNA binding sites, in agreement with the nuclear and cytoplasmic localization of the proteins. Computational and biochemical approaches defined the RNA recognition element (RRE) as a tandem CAC trinucleotide motif separated by a variable spacer region. Similar to other mRNA-binding proteins, RBPMS family of proteins relocalized to cytoplasmic stress granules under oxidative stress conditions suggestive of a support function for mRNA localization in large and/or multinucleated cells where it is preferentially expressed.

Keywords: PAR-CLIP; RBPMS; RNA recognition element; RNA-binding protein.

Figures

FIGURE 1.
FIGURE 1.
RBPMS overview. (A) RBPMS is conserved in vertebrates. Alignments were generated using ClustalW (NP_956553.1, NP_001002409.1, NP_082306.2, NP_919248.1, NP_001008710.1, NP_001036139, XP_003199078.1). (B) RBPMS and RBPMS2 localize to the nucleus and cytoplasm. Western Blotting analysis of nucleo-cytoplasmic fractions prepared from FLAG-HA RBPMS and RBPMS2 cell lines, as indicated. Nuclear (Nuc), cytoplasmic (Cyt), and total cell lysate (Tot) fractions were resolved on a 4%–12% SDS–polyacrylamide gel and then probed using an anti-HA antibody targeting FLAG-HA RBPMS and RBPMS2, and antibodies targeting ELAVL1 (HuR), TUBB (β-tubulin), and LMNB1 (lamin B) as controls for the purity of the cytoplasmic and nuclear fractions. (Right) Immunofluorescence staining of HA epitope in HEK293 cells stably expressing FLAG-HA RBPMS and FLAG-HA RBPMS2, using Hoechst and Phalloidin 546 as controls. (C) Phosphorimages of SDS-PAGE fractionating PAR-CLIP immunoprecipitate from constitutive and inducible overexpressing FLAG-HA-tagged RBPMS HEK293 cells. The cross-linked RNA–RBPMS complexes are indicated for two biological replicate experiments. Anti-HA Western-blotting control for expression and loading is shown at the bottom. Two protein bands were recognized by anti-HA antibody, one at the expected size based on recombinant full-length RBPMS and the other shorter, suggesting proteolytic cleavage (bands confirmed as RBPMS by mass spectrometry). (D) Overlap of PAR-CLIP clusters with ≥0.5 T-to-C conversion specificity between libraries A and B defines 6207 RBPMS-binding sites. (E) Genomic distribution of RBPMS-binding sites.
FIGURE 2.
FIGURE 2.
RBPMS and RBPMS2 expression during (A) mouse embryogenesis, (B) germ cell development, and (C) adult tissues. The mean affymetrix microarray expression data are depicted in log2 scale with bars showing SEM. The number of the biological replicates used in these analyses is shown in Supplemental Table 8. Expression of other genes with specific expression patterns during distinct developmental stages is also included. See Materials and Methods for description. (GV) germinal vesicles (immature oocytes); (MII) oocytes at metaphase II stage (mature oocytes); (Blast) blastocyst; (E) embryonic day; (ESC) embryonic stem cells; (M) male; (F) female; (GSC) germline stem cells (in vitro cultured postnatal testicular germline stem cells). Expression of all genes depicted on same plot for ease of representation.
FIGURE 3.
FIGURE 3.
RBPMS binds tandem CAC motifs separated by variable spacer length. (A) RBPMS RRE as identified by cERMIT analysis for library A and library B. (B) RSAT analysis defines CAC as a dyad motif with variable spacing. The Z-score of all trinucleotide dyads from each library is plotted; red triangles represent CAC dyads and adjacent number represents spacer oligonucleotide length; black dots represent all other trinucleotide dyads.
FIGURE 4.
FIGURE 4.
RBPMS and RBPMS2 proteins bind (AC)9 and (CAC)6 repeats in electrophoretic mobility shift assays (EMSA). (A) Synthetic RNAs representing 18-nt di- or trinucleotide repeats were radiolabeled (10 nM), incubated with 0–10 μM RBPMS recombinant protein (FLAG-HA-His6), and separated on 1% agarose gel. Binding conditions specified in Materials and Methods. The electrophoretic mobility shift observed for the (AAU)6 oligoribonucleotide (*) may not be due to RBPMS binding (see Results). We did not observe binding for (AG)9, (GGC)6, (GCC)6, A3G15, A18, C18, U18 with the same conditions (data not shown). (B) Binding specificity for recombinant RBPMS2 protein. Conditions same as in A. (C) Definition of minimal number of (AC)n repeats required for RBPMS binding. Conditions same as in A. (D) Coomassie-stained 15% SDS-PAGE indicates purity of recombinant RBPMS and RBPMS2 proteins.
FIGURE 5.
FIGURE 5.
RBPMS binds PAR-CLIP targets in EMSAs. (A) 21-nt synthetic RNAs representing clusters from mRNA targets identified by PAR-CLIP (NDUFA6, UBE2V1, SRM, and ETF1) were radiolabeled (10 nM), incubated with 0–10 μM recombinant RBPMS or RBPMS2 (FLAG-HA-His6), and separated on 1% agarose gel. The RNA sequences are shown on top, with nucleotides representing the RRE boxed. Residues in red illustrate point mutations of the synthetic RNAs. (B) Dissociation constant (Kd) determination for 21- and 15-nt synthetic RNAs representing the wild-type and mutant NDUFA6 cluster. (C) Binding study of RBPMS deletion proteins (His6) to synthetic (AC)9 RNA defines additional regions contributing to RNA binding. C-terminal deletion proteins are specified; N-terminal deletion protein (amino acids 21–196) required different binding conditions and could not be directly compared (data not shown).
FIGURE 6.
FIGURE 6.
RBPMS (AD, IL), G3BP1 (EH, MP), and poly(A) RNA localize to cytoplasmic granules after 400 μM arsenite administration. For assay conditions and description see Materials and Methods. RBPMS2 also localizes to cytoplasmic granules after arsenite administration (data not shown).

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