The Bin3 RNA methyltransferase targets 7SK RNA to control transcription and translation

Abstract

Bicoid-interacting protein 3 (Bin3) is a conserved RNA methyltransferase found in eukaryotes ranging from fission yeast to humans. It was originally discovered as a Bicoid (Bcd)-interacting protein in Drosophila, where it is required for anterior-posterior and dorso-ventral axis determination in the early embryo. The mammalian ortholog of Bin3 (BCDIN3), also known as methyl phosphate capping enzyme (MePCE), plays a key role in repressing transcription. In transcription, MePCE binds the non-coding 7SK RNA, which forms a scaffold for an RNA-protein complex that inhibits positive-acting transcription elongation factor b, an RNA polymerase II elongation factor. MePCE uses S-adenosyl methionine to transfer a methyl group onto the γ-phosphate of the 5' guanosine of 7SK RNA generating an unusual cap structure that protects 7SK RNA from degradation. Bin3/MePCE also has a role in translation regulation. Initial studies in Drosophila indicate that Bin3 targets 7SK RNA and stabilizes a distinct RNA-protein complex that assembles on the 3'-untranslated region of caudal mRNAs to prevent translation initiation. Much remains to be learned about Bin3/MeCPE function, including how it recognizes 7SK RNA, what other RNA substrates it might target, and how widespread a role it plays in gene regulation and embryonic development.

Figure 1

(A) Alignment of Bin3-related proteins from selected species. The conserved catalytic domain, AdoMet (SAM)-binding domain is indicated. We extended the region based on sequence similarities in these Bin3/MePCE orthologs to the indicated residues. (B) Sequence alignment of conserved AdoMet (SAM)-binding domain. Note the extended region present in the Drosophila protein that is absent in the other orthologs. Motif I, II, and III are generic motifs characteristic of all SAM-dependent methyltransferases including RNA, DNA and protein methyltransferases. Sequences are from D. melanogaster (NP_ 724468.1; aa786–1118), H. sapiens (NP_062552.2; aa412–689), M. musculus (NP_659162.3; aa389–666), C. elegans (NP_496573.1; aa116–370), A. thaliana (NP_568752; aa64–318) and S. pombe (NP_596220.1; aa3–261). The alignment was performed by using ClustalW (http://www.ch.embnet.org/software/ClustalW.html) with default parameters for all settings and formatted using GeneDoc (http://www.psc.edu/biomed/genedoc).

Figure 2

Structure of the mono 5′ γ-monomethyl guanosine triphosphate cap of 7SK RNA. The terminal and penultimate residues (GG) of 7SK RNA are shown with the methyl group added to the 5′ γ-phosphate (circled). This is added by Bin3/MePCE from using S-adenosyl-L-methionine as a donor. This cap structure is different than the canonical m⁷G cap of most eukaryotic mRNAs (see Box 1).

Figure 3

(A) Cartoon diagram of the X-ray crystal structure of the catalytic domain of human Bin3/MePCE (residues 431–685) bound to S-adenosyl-L-methionine (AdoMet) (Yellow). The structure was drawn with PDB coordinates 3G07 using PyMol. (B) Surface representation of human MePCE showing the locations of the consensus AdoMet-binding Motifs I (green), II (blue) and III (purple). (C) Active site of human MePCE highlighting hydrogen bond contacts (green dashed lines) between conserved MePCE residues and AdoMet. The position of the donor methyl group of AdoMet suggests that residues from Motifs II and III may be involved in orienting the 7SK RNA substrate for methylation. (D) Superposition of human MePCE and Drosophila Bin3. (E) The Drosophila structure (green) is a homology model based on based on the human structure and refined with the Swiss Model server. Residues modeled include aa806–864 and 981–1092. The modeled residues are 47% identical between human and Drosophila Bin-3. The RMS deviation for Cα atom positions is 3.5 Å. (F) Putative active site of Drosophila Bin3 based on homology modeling. Shown are residues that could potentially interact with AdoMet and RNA as in (C).

Figure 4

Secondary structure prediction of D. melanogaster 7SK RNA. Methods: Sfold software is based on structure ensemble sampling, structure clustering and centroid representation of clusters.^, It has been observed that the centroid structure of one of the clusters can often make an accurate structure prediction. However, the identity of the best performing centroid is unknown without additional information. For human 7SK RNA, the centroid structure for one of four structural clusters closely matched the proposed consensus structure. This indicated that, by using information from the consensus structure, we could identify the best performing centroid for structure prediction of 7SK RNA. For Drosophila 7SK RNA, we found that the centroid of one of two clusters closely resembles the consensus structure. This centroid is an informed predictor of the secondary structure for Drosophila 7SK RNA (Figure 4). The structure diagram was produced by the Sir_Graph program of the UNAFold package. Red = G-C; blue = A-U, green = G-U base pairing

Figure 5

Dynamic association of P-TEFb with the inhibitory 7SK snRNP. Figure summarizes work from a number of laboratories (reviewed in refs.^–). The P-TEFb transcription elongation factor is composed of CDK9 and CyclinT1 or T2. 7SK RNA forms a scaffold for an snRNP containing 7SK, LARP7, MePCE and hnRNPs. When P-TEFb, along with a HEXIM1/2 dimer is incorporated into the 7SK snRNP, it displaces hnRNPs and inactivates P-TEFb. P-TEFb inactivation involves interaction between HEXIMs and the cyclinT subunit of P-TEFb. 7SK RNA folding is different in the two complexes, as it is known to undergo structural rearrangements. LARP7 and HEXIMs are shown in the approximate positions they bind to 7SK RNA based on chemical protection experiments. See text for details. Figure is from Peterlin et al. (2011).

Figure 6

(A) Bin3/MePCE is required for translation repression in early development. Blastoderm-staged embryos (0–2 hr) are stained for Caudal protein (upper panels). Embryos and larvae are oriented with the anterior-left, dorsal-up. In wild-type embryos, Bicoid represses translation of caudal mRNA preventing accumulation of Caudal protein in the anterior (left panel). In bin3 loss-of-function mutant embryos, Bicoid is unable to repress caudal translation and Caudal protein accumulates throughout the embryo (right panel), resulting in failure to undergo proper head involution (lower, right panel), as visualized in first instar larvae. Data are from Singh et al. (2011). (B) Model for Bin3-Bicoid repression of caudal mRNA translation. Bin3 stabilizes Bicoid binding to the BRE in the caudal 3′ UTR. Bin3 does so by methylating and remaining bound to 7SK RNA which serve as scaffold for binding of other proteins, including the La-related protein, Larp1 and Ago2 and PABP which contribute to negative regulation of initiation. Although Bin3 is drawn as methylating the 5′ end of 7SK RNA as part of this repression complex, this modification may instead take place early, e.g. during transcription of 7SK RNA synthesis. See text for details. Model based on Singh et al. (2011).