Progress in 7SK ribonucleoprotein structural biology

Abstract

The 7SK ribonucleoprotein (RNP) is a dynamic and multifunctional regulator of RNA Polymerase II (RNAPII) transcription in metazoa. Comprised of the non-coding 7SK RNA, core proteins, and numerous accessory proteins, the most well-known 7SK RNP function is the sequestration and inactivation of the positive transcription elongation factor b (P-TEFb). More recently, 7SK RNP has been shown to regulate RNAPII transcription through P-TEFb-independent pathways. Due to its fundamental role in cellular function, dysregulation has been linked with human diseases including cancers, heart disease, developmental disorders, and viral infection. Significant advances in 7SK RNP structural biology have improved our understanding of 7SK RNP assembly and function. Here, we review progress in understanding the structural basis of 7SK RNA folding, biogenesis, and RNP assembly.

Keywords: RNA structural dynamics; RNA-protein interactions; X-ray crystallography; chemical probing; cryoEM; gene regulation; solution state NMR.

FIGURE 1

Timeline of advances in 7SK RNP structural biology and function.

FIGURE 2

Overview of 7SK RNP components involved in P-TEFb regulation. (A) Cartoon schematic of P-TEFb assembly and release. Gray, blue, and purple assemblies indicate proteins involved in 7SK RNA remodeling and P-TEFb release (Table 1). (B) Domain topologies of proteins required for P-TEFb assembly, with 7SK RNA and protein-protein interaction sites indicated. Domain and sequence information is for human 7SK RNA and proteins.

FIGURE 3

Secondary structure models of human 7SK RNA derived from biochemical and phylogenetic analysis. GAUC motifs are indicated by yellow circles. Residues are labeled according to evolutionary conservation (Gruber et al., 2008; Marz et al., 2009) and susceptibility to nuclease and/or chemical reactivity (Reddy et al., 1984; Wassarman and Steitz, 1991; Flynn et al., 2016; Olson et al., 2022). (A) Wassarman Steitz 7SK RNA secondary structure model (Wassarman and Steitz, 1991). (B) 7SK RNA secondary structure model from phylogenetic analysis (Marz et al., 2009). Inset legend: pink triangles show ssRNA-specific nuclease (RNase A1, RNase T1) cleavage activity in rat 7SK RNA (Reddy et al., 1984); red rectangles show conserved sequences in 7SK RNA (Gruber et al., 2008); yellow triangles show ssRNA-specific RNase T1 cleavage and chemical (DMS, Kethoxal and CMCT) modification in free 7SK RNA but not 7SK RNP; blue triangles show dsRNA-specific RNase V1 cleavage activity in free 7SK RNA but not 7SK RNP; filled purple circles show chemical modification sites in 7SK RNP but not free 7SK RNA; open purple circles show chemical modification sites in 7SK RNP (Wassarman and Steitz, 1991); filled cyan circles show in-cell SHAPE reactivity when BAF is bound to 7SK RNA; filled green circles show in-cell SHAPE reactivity when HEXIM1 is bound to the 7SK RNA (Flynn et al., 2016). (C,D) Secondary structure models of the 7SK RNA 5′ end in low and high MgCl₂ concentration (Brogie and Price, 2017). Line representations indicate regions with unreported secondary structure. Orange and pink circles show low and high SHAPE reactivity, respectively. (E,F) Consensus secondary structure models of major states A and B, respectively, from DANCE-Map studies (Olson et al., 2022). Orange and pink circles show moderate and high DMS reactivity, respectively.

FIGURE 4

Structures of 7SK RNA SL1 distal region. (A) Secondary structure model of the SL1 distal constructs used to determine solution NMR or X-ray crystal structures. N indicates variable apical loop sequence. ASMs are shown as defined by D’Souza and coworkers (Pham et al., 2018). (B) Base triples of arginine sandwich motifs (ASM) for solution NMR structure (PDB ID 7T1N). (C) Structures of SL1 distal constructs from solution NMR (PDB IDs 5IEM, 6MCI) or X-ray crystallography (PDB ID 5LYU).

FIGURE 5

Structures of MePCE and MePCE-RNA. (A) X-ray crystal structure of the MePCE MTase domain, (B) X-ray crystal structure of MePCE MTase bound to a capped 7SK SL1 proximal construct, (C) The 5′ RNA triphosphate is secured in the MePCE MTase active site by a “triphosphate binding tunnel” (PDB ID 6DCC) shown in surface and cartoon representation. Helices α0 and α7, which form on RNA binding, are colored blue. Cofactor byproduct SAH and RNA 5′ triphosphate, terminal base pair and 3′ overhang are shown in ball and stick representation. Methyl cap is colored cyan.

FIGURE 6

Structures of 7SK RNA 3′ end and Larp7 recognition of 7SK RNA. (A) Secondary structure model of the 7SK RNA 3′ end (nts 297–332), with Larp7 interaction sites indicated in brackets. (B) Solution NMR structure of 7SK SL4 bound to arginine (Arg). Arginine ligand is shown in stick representation. (C) X-ray crystal structure of Larp7 xRRM2 bound to SL4 upper stem. (D) X-ray crystal structure of Larp7 La module bound to UUU RNA sequence. (E) 7SK core RNP cryoEM structure (PDB ID 7SLQ) reveals cryptic β4 and α3 in Larp7 RRM1 and extensive La module-RNA binding surface.

FIGURE 7

CryoEM structures of 7SK core RNP ternary complex. RNA constructs model the (A) “linear” or (B) “circular” conformations. (C) SL4–Larp7–MePCE interface features extensive interactions (PDB ID 7SLQ). MePCE MTase is colored green and residues corresponding to helix α0, which becomes unstructured in the ternary complex, are colored blue. Larp7 RRM1 is colored apricot and helix α4 is colored maroon.

FIGURE 8

HEXIM1 and ARM recognition of 7SK SL1 RNA. (A) Solution NMR structures of SL1 distal bound to Tat or the first segment of the HEXIM1 ARM peptides. (B) ASM1-2 interacts with arginine residues 155–156 in HEXIM1 ARM (PDB ID 7T1N). (C) Solution NMR (PDB ID 2GD7) and X-ray crystal (PDB ID 3S9G) structures of HEXIM1 coiled-coil domain, with the putative CycT1 binding domain indicated in brackets.