Margaret L. Rodgers

Ribosomal subunits begin assembly during transcription of the ribosomal RNA (rRNA), when the rRNA begins to fold and associate with ribosomal proteins (RPs). In bacteria, the first steps of ribosome assembly depend upon recognition of the properly folded rRNA by primary assembly proteins such as S4, which nucleates assembly of the 16S 5′ domain. Recent evidence, however, suggests that initial recognition by S4 is delayed due to variable folding of the rRNA during transcription. Here, using single-molecule colocalization co-transcriptional assembly (smCoCoA), we show that the late-binding RP S12 specifically promotes the association of S4 with the pre-16S rRNA during transcription, thereby accelerating nucleation of 30S ribosome assembly. Order of addition experiments suggest that S12 helps chaperone the rRNA during transcription, particularly near the S4 binding site. S12 interacts transiently with the rRNA during transcription and, consequently, a high concentration is required for its chaperone activity. These results support a model in which late-binding RPs moonlight as RNA chaperones during transcription in order to facilitate rapid assembly.

Summary: Spliceosome activation is the process of creating the catalytic site for RNA splicing and occurs de novo on each intron following spliceosome assembly. Dozens of factors bind to or are released from the activating spliceosome including the Lsm2-8 heteroheptameric ring that binds the U6 small nuclear RNA (snRNA) 3’-end. Lsm2-8 must be released to permit active site stabilization by the Prp19-containing complex (NineTeen Complex, NTC); however, little is known about the temporal order of events and dynamic interactions that lead up to and follow Lsm2-8 release. We have used colocalization single molecule spectroscopy (CoSMoS) to visualize Lsm2-8 dynamics during activation of yeast spliceosomes. Lsm2-8 is recruited as a component of the tri-snRNP and is released after integration of the Prp19-containing complex (NineTeen Complex, NTC). Despite Lsm2-8 and the NTC being mutually exclusive in existing cryo-EM structures of yeast B complex spliceosomes, we identify a transient intermediate containing both and provide a kinetic framework for its formation and transformation during activation. Prior to assembly, the NTC rapidly and reversibly samples the spliceosome suggesting a mechanism for preventing NTC sequestration by defective spliceosomes that fail to properly activate. In complementary ensemble assays, we show that a base-pairing dependent ternary complex can form between Lsm2-8 and U2 and U6 helix II RNAs. Together our data suggest a Hfq-like function for Lsm2-8 in maintaining U2/U6 helix II integrity before it can be transferred to the NTC by transient formation of the spliceosome. Significance Statement: The spliceosome active site is created de novo during activation and involves numerous conformational and compositional changes. Here, we define a kinetic pathway for yeast spliceosome activation using single molecule fluorescence that includes transient intermediates not previously identified. Real-time measurements allow us to uncover rapid, reversible sampling interactions of the NineTeen Complex (NTC) that may prevent its accumulation on defective spliceosomes. By analogy with bacterial Hfq, we propose that the homologous Lsm2-8 proteins stabilize U2/U6 helix II during activation before the helix is transferred to the NTC in a short-lived spliceosome containing both Lsm2-8 and the NTC. Our data demonstrate how single molecule studies of activation can reveal kinetically-competent intermediates and complement cryo-EM studies of stalled or inhibited complexes.

The small nuclear RNA (snRNA) components of the spliceosome undergo many conformational rearrangements during its assembly, catalytic activation and disassembly. The U4 and U6 snRNAs are incorporated into the spliceosome as a base-paired complex within the U4/U6.U5 small nuclear ribonucleoprotein (tri-snRNP). U4 and U6 are then unwound in order for U6 to pair with U2 to form the spliceosome's active site. After splicing, U2/U6 is unwound and U6 annealed to U4 to reassemble the tri-snRNP. U6 rearrangements are crucial for spliceosome formation but are poorly understood. We have used single-molecule Förster resonance energy transfer and unwinding assays to identify interactions that promote U4/U6 unwinding and have studied their impact in yeast. We find that U4/U6 is efficiently unwound using DNA oligonucleotides by coupling unwinding of U4/U6 stem II with strand invasion of stem I. Unwinding is stimulated by the U6 telestem, which transiently forms in the intact U4/U6 RNA complex. Stabilization of the telestem in vivo results in accumulation of U4/U6 di-snRNP and impairs yeast growth. Our data reveal conserved mechanisms for U4/U6 unwinding and indicate telestem dynamics are critical for tri-snRNP assembly and stability.

The spliceosome is a complex machine composed of small nuclear ribonucleoproteins (snRNPs) and accessory proteins that excises introns from pre-mRNAs. After assembly the spliceosome is activated for catalysis by rearrangement of subunits to form an active site. How this rearrangement is coordinated is not well-understood. During activation, U4 must be released to allow U6 conformational change, while Prp19 complex (NTC) recruitment is essential for stabilizing the active site. We used multi-wavelength colocalization single molecule spectroscopy to directly observe the key events in Saccharomyces cerevisiae spliceosome activation. Following binding of the U4/U6.U5 tri-snRNP, the spliceosome either reverses assembly by discarding tri-snRNP or proceeds to activation by irreversible U4 loss. The major pathway for NTC recruitment occurs after U4 release. ATP stimulates both the competing U4 release and tri-snRNP discard processes. The data reveal the activation mechanism and show that overall splicing efficiency may be maintained through repeated rounds of disassembly and tri-snRNP reassociation.

The spliceosome undergoes dramatic changes in both small nuclear RNA (snRNA) composition and structure during assembly and pre-mRNA splicing. It has been previously proposed that the U2 snRNA adopts two conformations within the stem II region: stem IIa or stem IIc. Dynamic rearrangement of stem IIa into IIc and vice versa is necessary for proper progression of the spliceosome through assembly and catalysis. How this conformational transition is regulated is unclear; although, proteins such as Cus2p and the helicase Prp5p have been implicated in this process. We have used single-molecule Förster resonance energy transfer (smFRET) to study U2 stem II toggling between stem IIa and IIc. Structural interconversion of the RNA was spontaneous and did not require the presence of a helicase; however, both Mg²⁺ and Cus2p promote formation of stem IIa. Destabilization of stem IIa by a G53A mutation in the RNA promotes stem IIc formation and inhibits conformational switching of the RNA by both Mg²⁺ and Cus2p. Transitioning to stem IIa can be restored using Cus2p mutations that suppress G53A phenotypes in vivo. We propose that during spliceosome assembly, Cus2p and Mg²⁺ may work together to promote stem IIa formation. During catalysis the spliceosome could then toggle stem II with the aid of Mg²⁺ or with the use of functionally equivalent protein interactions. As noted in previous studies, the Mg²⁺ toggling we observe parallels previous observations of U2/U6 and Prp8p RNase H domain Mg²⁺-dependent conformational changes. Together these data suggest that multiple components of the spliceosome may have evolved to switch between conformations corresponding to open or closed active sites with the aid of metal and protein cofactors.

Large macromolecular complexes such as the spliceosomal small nuclear ribonucleoproteins (snRNPs) play a variety of roles within the cell. Despite their biological importance, biochemical studies of snRNPs and other machines are often thwarted by practical difficulties in the isolation of sufficient amounts of material. Studies of the snRNPs as well as other macromolecular machines would be greatly facilitated by new approaches that enable their isolation and biochemical characterization. One such approach is single-molecule pull-down (SiMPull) that combines in situ immunopurification of complexes from cell lysates with subsequent single-molecule fluorescence microscopy experiments. We report the development of a new method, called SNAP-SiMPull, that can readily be applied to studies of splicing factors and snRNPs isolated from whole-cell lysates. SNAP-SiMPull overcomes many of the limitations imposed by conventional SiMPull strategies that rely on fluorescent proteins. We have used SNAP-SiMPull to study the yeast branchpoint bridging protein (BBP) as well as the U1 and U6 snRNPs. SNAP-SiMPull will likely find broad use for rapidly isolating complex cellular machines for single-molecule fluorescence colocalization experiments.