A threefold RNA-protein interface in the signal recognition particle gates native complex assembly

Abstract

Intermediate states play well-established roles in the folding and misfolding reactions of individual RNA and protein molecules. In contrast, the roles of transient structural intermediates in multi-component ribonucleoprotein (RNP) assembly processes and their potential for misassembly are largely unexplored. The SRP19 protein is unstructured but forms a compact core domain and two extended RNA-binding loops upon binding the signal recognition particle (SRP) RNA. The SRP54 protein subsequently binds to the fully assembled SRP19-RNA complex to form an intimate threefold interface with both SRP19 and the RNA and without significantly altering the structure of SRP19. We show, however, that the presence of SRP54 during SRP19-RNA assembly dramatically alters the folding energy landscape to create a non-native folding pathway that leads to an aberrant SRP19-RNA conformation. The misassembled complex arises from the surprising ability of SRP54 to bind rapidly to an SRP19-RNA assembly intermediate and to interfere with subsequent folding of one of the RNA binding loops at the three-way protein-RNA interface. An incorrect temporal order of assembly thus readily yields a non-native three-component ribonucleoprotein particle. We propose there may exist a general requirement to regulate the order of interaction in multi-component RNP assembly reactions by spatial or temporal compartmentalization of individual constituents in the cell.

Figure 1

The signal recognition particle (SRP). (A) Architecture of the mammalian SRP. SRP proteins are shown as colored and gray ovals; the RNA is yellow. (B) ‘SRP54-late’ model for cellular assembly of the mammalian SRP. Five of six SRP proteins (SRP9, SRP14, SRP19, SRP68, and SRP72) (gray and green ovals) enter the nucleus or nucleolus to assemble with the SRP RNA. The partially assembled complex is then transported back into the cytoplasm to bind SRP54 (purple) and form the native SRP holocomplex. (C) In vitro scheme for native SRP19-SRP54-SRP RNA ternary complex formation via the SRP54-late assembly pathway. (D) ‘SRP54-early’ pathway that leads to formation of a non-native ternary complex, termed the non-compartmentalized complex, in which two RNA binding loops in SRP19 do not fold to their native conformation.10 (E) Three-fold protein-RNA interface in the native SRP ternary complex. SRP19 loop 2 (gray) is positioned in a cleft formed by SRP54 and the RNA. The figure shows the same complex as panel C, but has been rotated ~90° to afford a view from the ‘top’ of the three-component interface.

Figure 2

SRP19 and the large subunit SRP RNA (LS RNA). (A) SRP19 structural motifs when bound to the SRP RNA. SRP core is green and the two RNA binding loops are gray. Sites of fluorophore attachment are shown as spheres. (B) Alexa 555 derivatized RNA:DNA hybrid. RNA and DNA are shown in black and gray, respectively. (C) LS RNA.

Figure 3

FRET-based analysis of SRP assembly. (A) Scheme for monitoring SRP assembly using Alexa 488-derivatized SRP19 (green) and the Alexa 555-LS RNA (yellow and gray). SRP54 (purple) binding is detected by its affect on the SRP19-RNA complex. (B, C) Three distinct assembly steps are detected during native SRP19-SRP54-SRP RNA ternary complex formation. (1) A rapid burst phase quenching of fluorescence during initial SRP19-RNA assembly (green). (2) Well-resolved increase or decrease in fluorescence emission intensity as the SRP19-RNA complex matures to the native structure (green). (3) An increase in fluorescence intensity due to SRP54 binding to the pre-formed SRP19-RNA complex (purple). Free Alexa 647 reference fluorophore is gray.

Figure 4

Burst phase assembly step corresponds to Encounter complex formation between SRP19 and the RNA. (A,B) SRP19-RNA assembly was monitored for the native sequence RNA or an A149U mutant that cannot form native RNA-RNA interactions (solid and open symbols, respectively). Arrows indicate burst phases observed with both RNAs. (C) Addition of SRP54 has no effect on the Encounter complex formed between SRP19 and the A149U RNA.

Figure 5

Conformational rearrangements at distinct SRP19 structural motifs during RNP assembly. (A) Scheme for monitoring SRP19-RNA assembly using single Alexa 488 fluorophore experiments. (B) Fluorescence-detected assembly at the RNA-binding loops (open symbols) proceeds significantly faster than assembly of the SRP19 core domain (solid symbols).

Figure 6

SRP19 structural motifs assemble with the RNA via distinct mechanisms. (A–D) Rate of change in fluorescence intensity as a function of RNA concentration for SRP19 variants. Slopes (dashed lines) yield second order rate constants for assembly as detected in distinctive SRP19 loop and core structural motifs. Most experiments were performed with Alexa 488-labeled SRP19 (squares). For the 72Cys and 106Cys variants, assembly was also monitored using the BODIBY-FL fluorophore (circles).

Figure 7

One step assembly of SRP54 to the pre-formed SRP19-RNA complex. (A, B) The rate constant for SRP54 binding to the pre-formed Alexa 488-labeled SRP19-RNA complex was obtained by fitting the change in fluorescence intensity over time to a second order rate equation. (C,D) All SRP54 concentrations yielded identical second-order rate constants.

Figure 8

The extent of non-compartmentalized complex formation is concentration dependent. (A) Scheme for monitoring native vs. non-compartmentalized assembly using single fluorophore experiments. Native assembly (the SRP54-late pathway) involves ordered binding by SRP19 (green traces) and SRP54 (purple traces). For non-compartmentalized assembly (the SRP54-early pathway), both proteins assemble simultaneously (black traces). (B,C) Visualization of native and non-compartmentalized ternary complex formation using single fluorophore experiments. Ratios of SRP19, SRP54 and RNA components were held constant at 1:1:2 in all experiments; protein concentrations are given for each panel. Non-compartmentalized complex formation is reported directly as the intensity difference between purple and black traces (see red brackets). (D) Expected concentration dependence for SRP54 binding post-Encounter versus post-Stable complex formation. k₁ is a compound rate constant reflecting formation of the Stable complex via the kinetically linked Encounter complex. Dashed arrows show putative, concentration-dependent (k′₃[ ]), steps for SRP54-mediated misassembly.

Figure 9

Mechanisms for native and non-compartmentalized SRP assembly. (A) Native and non-compartmentalized assembly pathways are gated at the Stable complex. (B,C) Folding energy landscapes for SRP19 during SRP ternary complex formation via SRP54-late and SRP54-early pathways.