RNA-binding is an ancient trait of the Annexin family

Abstract

Introduction: The regulation of intracellular functions in mammalian cells involves close coordination of cellular processes. During recent years it has become evident that the sorting, trafficking and distribution of transport vesicles and mRNA granules/complexes are closely coordinated to ensure effective simultaneous handling of all components required for a specific function, thereby minimizing the use of cellular energy. Identification of proteins acting at the crossroads of such coordinated transport events will ultimately provide mechanistic details of the processes. Annexins are multifunctional proteins involved in a variety of cellular processes associated with Ca²⁺-regulation and lipid binding, linked to the operation of both the endocytic and exocytic pathways. Furthermore, certain Annexins have been implicated in the regulation of mRNA transport and translation. Since Annexin A2 binds specific mRNAs via its core structure and is also present in mRNP complexes, we speculated whether direct association with RNA could be a common property of the mammalian Annexin family sharing a highly similar core structure. Methods and results: Therefore, we performed spot blot and UV-crosslinking experiments to assess the mRNA binding abilities of the different Annexins, using annexin A2 and c-myc 3'UTRs as well as c-myc 5'UTR as baits. We supplemented the data with immunoblot detection of selected Annexins in mRNP complexes derived from the neuroendocrine rat PC12 cells. Furthermore, biolayer interferometry was used to determine the K_D of selected Annexin-RNA interactions, which indicated distinct affinities. Amongst these Annexins, Annexin A13 and the core structures of Annexin A7, Annexin A11 bind c-myc 3'UTR with K_Ds in the nanomolar range. Of the selected Annexins, only Annexin A2 binds the c-myc 5'UTR indicating some selectivity. Discussion: The oldest members of the mammalian Annexin family share the ability to associate with RNA, suggesting that RNA-binding is an ancient trait of this protein family. Thus, the combined RNA- and lipid-binding properties of the Annexins make them attractive candidates to participate in coordinated long-distance transport of membrane vesicles and mRNAs regulated by Ca²⁺. The present screening results can thus pave the way for studies of the multifunctional Annexins in a novel cellular context.

Keywords: 3′untranslated region; Annexin; RNA granules; RNA-binding; c-Myc; mRNA; mRNP complexes.

FIGURE 1

AnxA1, AnxA2, AnxA4, AnxA5, AnxA6, AnxA7, AnxA10, AnxA11 and AnxA13 present in the cytoskeleton fraction (Panel A) are associated with non-polysomal mRNP complexes (Panel B) of PC12 cells. Panel (A) 30 µg of the cytoskeletal fraction (lane 1) and cytoskeleton-bound polysomes (lane 2) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Panel (B) samples prepared from oligo (dT)-bound mRNP complexes from the cytoskeletal fraction [supernatant after centrifugation for 2 h 100,000 g above a 1 M (35%) sucrose cushion] (lanes 3 and 4), without (lane 3) or with RNase (lane 4) treatment, as indicated above the Western blots, were subjected to similar analysis. The blots were probed with antibodies against the different Anxs and against PABP1 as a marker for poly(A)-containing mRNAs, as indicated. Antibodies against the ribosomal subunit S6 were used to inform of the distribution of ribosomes. In addition, the blots were probed with antibodies against early endosomes (EEA1), late endosomes (Rab7) and recycling endosomes (Rab11). SPC25 and LAMP1 were not detectable in any of the fractions (results not shown). Visualization of the immunoreactive protein bands was performed using the ChemiDocTM XRS+ molecular imager after incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies and enhanced chemiluminescence (ECL)-reagent. The blots shown are representative for results from three experiments.

FIGURE 2

The binding of rat Anxs to total RNA, total poly(A)-containing mRNAs and specific RNAs. Panel (A) shows schematically the protocol for the spotting of proteins on membranes to ease the evaluation of the data. Recombinant rat AnxA1-to-AnxA11, AnxA13 and BSA were spotted on nitrocellulose membranes in 3 μL samples containing 5, 10 or 20 μM protein, as indicated in Panel (A). Subsequently, the membranes were incubated for 30 min with 200,000 cpm of the following probes: [³²P]-labeled total RNA [Panel (B)] or [³²P]-labeled poly(A) -containing total mRNA [Panel (C)], in vitro transcribed [³²P] rUTP-labeled anxA2 mRNA Panel (D), c-myc 3′UTR [Panel (E)], or the coding region of hRLuc Panel (F)]. All incubations were carried out in RNA-binding buffer in the presence of 100 μM Ca²⁺ and 1 μg/μL tRNA to block unspecific binding of RNA. Heat-denatured AnxA2 and BSA were spotted as negative controls since they do not bind RNA. The radioactive signals were detected after 48 h exposure of the spot blots to a radiosensitive screen in a phosphor-imager (Fuji Bas-5000). The dark signals (spots) on the membrane indicate RNA binding to the indicated recombinant rat Anx proteins. Panel (G) Samples of Anxs as indicated were subjected to 4%–15% SDS-PAGE and the proteins were stained with Coomassie Brilliant Blue. Standard proteins (PageRuler prestained protein ladder) are indicated to the left. The spot blots are representative blots from 3 experiments.

FIGURE 3

UV-crosslinking competition experiments employing rat Anxs and radiolabeled [α³²P]-rUTP c-myc or anxA2 3′UTRs. 2 μM purified rat Anxs (except 0.4 µM of AnxA7; 0.3 µM of AnxA9 and 0.5 µM of AnxA11) were UV-crosslinked in the absence of RNA (lanes 1 and 5). 100,000 cpm of radiolabeled anxA2 3′UTR (7 fmoles) or c-myc 3′UTR (6 fmoles) were UV-crosslinked to purified rat Anxs in the absence (lanes 2 and 6) or presence of 25x (lanes 3 and 7), or 50x (lanes 4 and 8) molar excess of the corresponding unlabeled transcript. 2 μM BSA and mutant AnxA2 served as negative controls. After UV-crosslinking and RNase treatment, the samples were subjected to 4%–15% SDS-PAGE and the proteins were stained with Coomassie Brilliant Blue (lanes 1–4), whereafter the gels were dried. The [α³²P]-rUTP-labeled RNA covalently bound to the respective Anxs, as indicated, was visualized using screens and phosphor-imaging following an overnight (c-myc 3′UTR) or 6 h (anxA2 3′UTR) exposure (lanes 5–8). PageRuler prestained protein ladder (from top to bottom: 100 kDa, 70 kDa (the most prominent band), 55 kDa, 40 kDa, 35 kDa and 25 kDa) are shown to the left of the AnxA1, AnxA3, AnxA5, AnxA7, AnxA8, AnxA10, Δ188AnxA11 and BSA samples. The representative images are from two experiments performed with competition while RNA-Anx binding was performed four times without competition.

FIGURE 4

Alignment of the RNA-binding site in domain IV of AnxA2 (indicated by the red box) with corresponding sites in the other rat Anxs. (A) In the “RNA-binding site”, positively charged amino acid residues are labeled green and polar residues purple. X above the sequences indicates the amino acid residues involved in RNA-binding in AnxA2. (B) WebLogo representation (Schneider and Stephens, 1990; Crooks et al., 2004) of the alignment of the RNA-binding site in AnxA2 shown inside the red box in Panel A. The overall height of a stack indicates the sequence conservation at the position of that amino acid residue among the 13 different sequences, while the height of symbols within the stack indicates the relative frequencies of the various amino acid residues found at that position in the RNA-binding site.