Phosphorylation at intrinsically disordered regions of PAM2 motif-containing proteins modulates their interactions with PABPC1 and influences mRNA fate.

Cytoplasmic poly(A)-binding protein (PABP) C1 recruits different interacting partners to regulate mRNA fate. The majority of PABP-interacting proteins contain a PAM2 motif to mediate their interactions with PABPC1. However, little is known about the regulation of these interactions or the corresponding functional consequences. Through in silico analysis, we found that PAM2 motifs are generally embedded within an extended intrinsic disorder region (IDR) and are located next to cluster(s) of potential serine (Ser) or threonine (Thr) phosphorylation sites within the IDR. We hypothesized that phosphorylation at these Ser/Thr sites regulates the interactions between PAM2-containing proteins and PABPC1. In the present study, we have tested this hypothesis using complementary approaches to increase or decrease phosphorylation. The results indicate that changing the extent of phosphorylation of three PAM2-containing proteins (Tob2, Pan3, and Tnrc6c) alters their ability to interact with PABPC1. Results from experiments using phospho-blocking or phosphomimetic mutants in PAM2-containing proteins further support our hypothesis. Moreover, the phosphomimetic mutations appreciably affected the functions of these proteins in mRNA turnover and gene silencing. Taken together, these results provide a new framework for understanding the roles of intrinsically disordered proteins in the dynamic and signal-dependent control of cytoplasmic mRNA functions.

Protein roles in group I intron RNA folding: the tyrosyl-tRNA synthetase CYT-18 stabilizes the native state relative to a long-lived misfolded structure without compromising folding kinetics.

The Neurospora crassa CYT-18 protein is a mitochondrial tyrosyl-tRNA synthetase that also promotes self-splicing of group I intron RNAs by stabilizing the functional structure in the conserved core. CYT-18 binds the core along the same surface as a common peripheral element, P5abc, suggesting that CYT-18 can replace P5abc functionally. In addition to stabilizing structure generally, P5abc stabilizes the native conformation of the Tetrahymena group I intron relative to a globally similar misfolded conformation that has only local differences within the core and is populated significantly at equilibrium by a ribozyme variant lacking P5abc (E(DeltaP5abc)). Here, we show that CYT-18 specifically promotes formation of the native group I intron core from this misfolded conformation. Catalytic activity assays demonstrate that CYT-18 shifts the equilibrium of E(DeltaP5abc) toward the native state by at least 35-fold, and binding assays suggest an even larger effect. Thus, similar to P5abc, CYT-18 preferentially recognizes the native core, despite the global similarity of the misfolded core and despite forming crudely similar complexes, as revealed by dimethyl sulfate footprinting. Interestingly, the effects of CYT-18 and P5abc on folding kinetics differ. Whereas P5abc inhibits refolding of the misfolded conformation by forming peripheral contacts that must break during refolding, CYT-18 does not display analogous inhibition, most likely because it relies to a greater extent on direct interactions with the core. Although CYT-18 does not encounter this RNA in vivo, our results suggest that it stabilizes its cognate group I introns relative to analogous misfolded intermediates. By specifically recognizing native structural features, CYT-18 may also interact with earlier folding intermediates to avoid RNA misfolding or to trap native contacts as they form. More generally, our results highlight the ability of a protein cofactor to stabilize a functional RNA structure specifically without incurring associated costs in RNA folding kinetics.

Mapping of the functional boundaries and secondary structure of the mouse mammary tumor virus Rem-responsive element.

Mouse mammary tumor virus (MMTV) is a complex retrovirus that encodes at least three regulatory and accessory proteins, including Rem. Rem is required for nuclear export of unspliced viral RNA and efficient expression of viral proteins. Our previous data indicated that sequences at the envelope-3' long terminal repeat junction are required for proper export of viral RNA. To further map the Rem-responsive element (RmRE), reporter vectors containing various portions of the viral envelope gene and the 3' long terminal repeat were tested in the presence and absence of Rem in transient transfection assays. A 476-bp fragment that spans the envelope-long terminal repeat junction had activity equivalent to the entire 3'-end of the mouse mammary tumor virus genome, but further deletions at the 5'- or 3'-ends reduced Rem responsiveness. RNase structure mapping of the full-length RmRE and a 3'-truncation suggested multiple domains with local base pairing and intervening single-stranded segments. A secondary structure model constrained by these data is reminiscent of the RNA response elements of other complex retroviruses, with numerous local stem-loops and long-range base pairs near the 5'- and 3'-boundaries, and differs substantially from an earlier model generated without experimental constraints. Covariation analysis provides limited support for basic features of our model. Reporter assays in human and mouse cell lines revealed similar boundaries, suggesting that the RmRE does not require cell type-specific proteins to form a functional structure.

Deletion of the P5abc peripheral element accelerates early and late folding steps of the Tetrahymena group I ribozyme.

The P5abc peripheral element stabilizes the Tetrahymena group I ribozyme and enhances its catalytic activity. Despite its beneficial effects on the native structure, prior studies have shown that early formation of P5abc structure during folding can slow later folding steps. Here we use a P5abc deletion variant E(deltaP5abc) to systematically probe the role of P5abc throughout tertiary folding. Time-resolved hydroxyl radical footprinting shows that E(deltaP5abc) forms its earliest stable tertiary structure on the millisecond time scale, approximately 5-fold faster than the wild-type ribozyme, and stable structure spreads throughout E(deltaP5abc) in seconds. Nevertheless, activity measurements show that the earliest detectable formation of native E(deltaP5abc) ribozyme is much slower (approximately 0.6 min(-1)), in a manner similar to that of the wild type. Also similar, only a small fraction of E(deltaP5abc) attains the native state on this time scale under standard conditions at 25 degrees C, whereas the remainder misfolds; footprinting experiments show that the misfolded conformer shares structural features with the long-lived misfolded conformer of the wild-type ribozyme. Thus, P5abc does not have a large overall effect on the rate-limiting step(s) along this pathway. However, once misfolded, E(deltaP5abc) refolds to the native state 80-fold faster than the wild-type ribozyme and is less accelerated by urea, indicating that P5abc stabilizes the misfolded structure relative to the less-ordered transition state for refolding. Together, the results suggest that, under these conditions, even the earliest tertiary folding intermediates of the wild-type ribozyme represent misfolded species and that P5abc is principally a liability during the tertiary folding process.

Structural specificity conferred by a group I RNA peripheral element.

Like proteins, structured RNAs must specify a native conformation that is more stable than all other possible conformations. Local structure is much more stable for RNA than for protein, so it is likely that the principal challenge for RNA is to stabilize the native structure relative to misfolded and partially folded intermediates rather than unfolded structures. Many structured RNAs contain peripheral structural elements, which surround the core elements. Although it is clear that peripheral elements stabilize structure within RNAs that contain them, it has not yet been explored whether they specifically stabilize the native states relative to alternative folds. A two-piece version of the group I intron RNA from Tetrahymena is used here to show that the peripheral element P5abc binds to the native conformation of the rest of the RNA 50,000 times more tightly than it binds to a long-lived misfolded conformation. Thus, P5abc stabilizes the native conformation by approximately 6 kcal/mol relative to this misfolded conformation. Further, activity measurements show that for the RNA lacking P5abc, the native conformation is only marginally preferred over the misfolded conformation (<0.5 kcal/mol), indicating that the peripheral structure of this RNA is required to achieve a significant thermodynamic preference for the native state. Such "structural specificity" may be a general function of RNA peripheral domains.