Importance of the NCp7-like domain in the recognition of pre-let-7g by the pluripotency factor Lin28

Abstract

The pluripotency factor Lin28 is a highly conserved protein comprising a unique combination of RNA-binding motifs, an N-terminal cold-shock domain and a C-terminal region containing two retroviral-type CCHC zinc-binding domains. An important function of Lin28 is to inhibit the biogenesis of the let-7 family of microRNAs through a direct interaction with let-7 precursors. Here, we systematically characterize the determinants of the interaction between Lin28 and pre-let-7 g by investigating the effect of protein and RNA mutations on in vitro binding. We determine that Lin28 binds with high affinity to the extended loop of pre-let-7 g and that its C-terminal domain contributes predominantly to the affinity of this interaction. We uncover remarkable similarities between this C-terminal domain and the NCp7 protein of HIV-1, not only in terms of primary structure but also in their modes of RNA binding. This NCp7-like domain of Lin28 recognizes a G-rich bulge within pre-let-7 g, which is adjacent to one of the Dicer cleavage sites. We hypothesize that the NCp7-like domain initiates RNA binding and partially unfolds the RNA. This partial unfolding would then enable multiple copies of Lin28 to bind the extended loop of pre-let-7 g and protect the RNA from cleavage by the pre-microRNA processing enzyme Dicer.

Figure 1.

The Lin28 protein, pre-let-7g RNA and related sequences used in this study. (A) Schematic representation of the primary structures of Lin28 and deletion fragments. The gray boxes delineate sequences of known RNA-binding motifs: a cold shock domain (CSD) and a pair of retroviral–type CCHC zinc-binding domains (ZBD1 and ZBD2). (B) Schematic representation of pre-let-7g, indicating the regions (gray boxes) from which TL-let-7g and duplex let-7g GNRA were derived. (C and D) Primary and secondary structures of the (C) duplex let-7g GNRA and (D) TL-let-7g. Nucleotides within the mature miRNA sequence are in blue and non-natural nucleotides are shown in lowercase. In (D), site-specific mutations of TL-let-7g are in red and regions that were replaced by alternative structured elements are boxed.

Figure 2.

EMSA of TL-let-7g with Lin28_1–209 and Lin28_119–180. (A) Typical EMSA performed with 1 pM of 5′-[³²P]-labeled TL-let-7g and increasing concentrations of Lin28_1–209 (0.0, 0.002, 0.010, 0.025, 0.050, 0.075, 0.10, 0.15, 0.20, 0.50, 1.0, 2.5 and 5.0 nM). (B) Typical EMSA for TL-let-7g and increasing concentrations of Lin28_119–180 (0.0, 0.02, 0.10, 0.25, 0.50, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 5.0,10, 25 and 50 nM). (C) The bound fraction of RNA is plotted against the total concentration of protein. The data for binding of TL-let-7g to Lin28_1–209 (squares) are fitted to both the one site binding equation (red line; K_d = 0.2 nM) and the Hill equation (blue line; K_d = 0.13 nM). The data for binding of TL-let-7g to Lin28_119–180 (dots) are fitted to the one site binding equation (green line; K_d = 1.3 nM).

Figure 3.

Sequence similarity between the HIV-1 NCp7 and the C-terminal domain of Lin28. The sequences of HIV-1 NCp7 and Lin28 from Mus musculus (mmu), Homo sapiens (hsa), Gallus gallus (gga), Xenopus laevis (xla) and Danio rerio (dre) were aligned using ClustalW2 (70). A consensus sequence is given with the standard one-letter code in capital letters for amino acids, as well as the following notation: a, aromatic; h, hydrophobic; p, polar; +, positively charged. The schematic representation of NCp7 highlights the domains that contribute to RNA binding: an N-terminal KR-rich domain and two zinc-binding domains (ZBD1 and ZBD2). The residues of NCp7 in red and blue make direct contact with zinc and RNA, respectively (33,54). Those residues that could play an equivalent role in Lin28 are similarly colored.

Figure 4.

(A) Superposition of 2D ¹H-¹⁵N HSQC spectra of 1 mM ¹³C/¹⁵N-labeled Lin28_119–180 in the free form (black) and bound to 1 mM TL-let-7g (red). The signals from the free form that display a significant chemical shift change (Δδ > 0.4 ppm) as a result of RNA binding are annotated and the change is illustrated with an arrow. A very weak signal for D137 in the complex is indicated by a star. (B) Histogram displaying the differences in chemical shifts (Δδ in ppm ± 0.03 ppm) observed after the addition of a molar equivalent of TL-let-7g to 1 mM ¹³C/¹⁵N-labeled Lin28_119–180. The chemical shift differences (Δδ) were calculated according to the formula Δδ = [(ΔH^N)²+ (0.17ΔN^H)²]^1/2.

Figure 5.

Footprint analysis of TL-let-7g with RNase T₁. (A) Secondary structure of TL-let-7g with the mapping of in-line probing and T₁ footprinting data. Residues that are the most susceptible to spontaneous cleavage through in-line attack are in bold (Supplementary Figure S2), and residues that experience a significant reduction (I_p/I₀ = −4) or enhancement (I_p/I₀ = +4) of T₁ cleavage in the presence of Lin28_119–180 are shaded in red and blue, respectively. (B) Typical RNA footprinting gel of TL-let-7g in the absence and presence of Lin28_119–180 (at concentrations of 0, 1, 5, 25, 100, 250, 500 and 1000 nM). Lanes with input TL-let-7g (RNA), an alkaline hydrolysis ladder (OH−) and a T₁ hydrolysis ladder (T1) are also included. (C) Histogram of normalized band sensitivity (I_p/I_0, where I_p and I₀ are, respectively, the intensity in the presence and absence of protein) for T₁ cleaveage of each guanine obtained at 25–500 nM Lin28_119–180.