Eukaryotes possess eight highly conserved Lsm (like Sm) proteins that assemble into circular, heteroheptameric complexes, bind RNA, and direct a diverse range of biological processes. Among the many essential functions of Lsm proteins, the cytoplasmic Lsm1-7 complex initiates mRNA decay, while the nuclear Lsm2-8 complex acts as a chaperone for U6 spliceosomal RNA. It has been unclear how these complexes perform their distinct functions while differing by only one out of seven subunits. Here, we elucidate the molecular basis for Lsm-RNA recognition and present four high-resolution structures of Lsm complexes bound to RNAs. The structures of Lsm2-8 bound to RNA identify the unique 2′,3′ cyclic phosphate end of U6 as a prime determinant of specificity. In contrast, the Lsm1-7 complex strongly discriminates against cyclic phosphates and tightly binds to oligouridylate tracts with terminal purines. Lsm5 uniquely recognizes purine bases, explaining its divergent sequence relative to other Lsm subunits. Lsm1-7 loads onto RNA from the 3′ end and removal of the Lsm1 C-terminal region allows Lsm1-7 to scan along RNA, suggesting a gated mechanism for accessing internal binding sites. These data reveal the molecular basis for RNA binding by Lsm proteins, a fundamental step in the formation of molecular assemblies that are central to eukaryotic mRNA metabolism.
There are few methods available for the rapid discovery of multi-target drugs. Herein, we describe the template-assisted, target-guided discovery of small molecules that recognize d(CTG) in the expanded d(CTG•CAG) sequence and its r(CUG) transcript that cause myotonic dystrophy type 1 (DM1). A positive cross-selection was performed using a small library of 30 monomeric alkyneand azide-containing ligands capable of producing more than 5000 possible di-and trimeric click products. The monomers were incubated with d(CTG) 16 or r(CUG) 16 under physiological conditions and both sequences showed selectivity in the proximity-accelerated azide-alkyne [3+2]cycloaddition click reaction. The limited number of click products formed in both selections and the even smaller number of common products suggests that this method is a useful tool for the discovery of single-target and multi-target lead therapeutic agents.
U6 snRNA undergoes post-transcriptional 3′ end modification prior to incorporation into the active site of spliceosomes. The responsible exoribonuclease is Usb1, which removes nucleotides from the 3′ end of U6 and, in humans, leaves a 2′,3′ cyclic phosphate that is recognized by the Lsm2–8 complex. Saccharomycescerevisiae Usb1 has additional 2′,3′ cyclic phosphodiesterase (CPDase) activity, which converts the cyclic phosphate into a 3′ phosphate group. Here we investigate the molecular basis for the evolution of Usb1 CPDase activity. We examine the structure and function of Usb1 from Kluyveromyces marxianus, which shares 25 and 19% sequence identity to the S. cerevisiae and Homo sapiens orthologs of Usb1, respectively. We show that K. marxianus Usb1 enzyme has CPDase activity and determined its structure, free and bound to the substrate analog uridine 5′-monophosphate. We find that the origin of CPDase activity is related to a loop structure that is conserved in yeast and forms a distinct penultimate (n – 1) nucleotide binding site. These data provide structural and mechanistic insight into the evolutionary divergence of Usb1 catalysis.
Eukaryotes possess eight highly conserved Lsm (like Sm) proteins that assemble into circular, heteroheptameric complexes, bind RNA, and direct a diverse range of biological processes. Among the many essential functions of Lsm proteins, the cytoplasmic Lsm1-7 complex initiates mRNA decay, while the nuclear Lsm2-8 complex acts as a chaperone for U6 spliceosomal RNA. It has been unclear how these complexes perform their distinct functions while differing by only one out of seven subunits. Here, we elucidate the molecular basis for Lsm-RNA recognition and present four high-resolution structures of Lsm complexes bound to RNAs. The structures of Lsm2-8 bound to RNA identify the unique 2′,3′ cyclic phosphate end of U6 as a prime determinant of specificity. In contrast, the Lsm1-7 complex strongly discriminates against cyclic phosphates and tightly binds to oligouridylate tracts with terminal purines. Lsm5 uniquely recognizes purine bases, explaining its divergent sequence relative to other Lsm subunits. Lsm1-7 loads onto RNA from the 3′ end and removal of the Lsm1 C-terminal region allows Lsm1-7 to scan along RNA, suggesting a gated mechanism for accessing internal binding sites. These data reveal the molecular basis for RNA binding by Lsm proteins, a fundamental step in the formation of molecular assemblies that are central to eukaryotic mRNA metabolism.
Lsm proteins form heptameric rings that bind RNA. Eukaryotes contain Lsm1–7 and Lsm2–8 ring structures, which are comprised of Lsm2, Lsm3, Lsm4, Lsm5, Lsm6, Lsm7 and either Lsm1 or Lsm8. Thus, the two rings differ by only one component. This small change in composition has a profound effect on the biological function of the two rings. Lsm1–7 is located in the cytosol and plays an essential role in the decay of messenger RNA, whereas Lsm2–8 is located in the nucleus and specifically binds to the 3′ end of U6 RNA. It is important to understand the RNA binding properties of Lsm rings. For example, the Lsm1–7 mRNA decay pathway regulates a large proportion of gene expression (Garneau et al., 2007).It is known that Lsm2–8 binds to U6 snRNA via a 3′ polyuridine stretch and a terminal phosphate group (Licht et al., 2008; Didychuk et al., 2017). In contrast, the RNA binding properties of the Lsm1–7 ring are not well understood. Free Lsm1–7 rings in S. cerevisiae lack tight binding affinity for polyuridine tracts (Zhou and Zhou, et al., 2014). The cognate RNA sequence for the free Lsm1–7 ring is unknown, although association of the ring with the Pat1 protein can stimulate binding of the ring to polyuridine tracts (Wu et al., 2014). Crystal structures of the free Lsm1–7 complex from S. cerevisiae show that the C‐terminal region of Lsm1 impinges on the uridine‐phosphate binding pocket observed in the S. cerevisiae Lsm2–8 ring with U6 snRNA (Zhou and Zhou, et al., 2014; Sharif and Conti, 2013; Montemayor et al., 2018). We hypothesized that Lsm1 is a central regulator of RNA binding activity in the Lsm1–7 complex and that Pat1 is an allosteric regulator of Lsm1.We have purified Lsm rings from the fission yeast S. pombe, which closely resemble humans with respect to Lsm ring sequence and known binding properties. We have made several novel observations. First, free Lsm1–7 is capable of binding polyuridine tracts in the absence of the Pat1 cofactor if the polyuridine tract is followed by an adenosine residue. This is the first observation of tight RNA binding affinity for Lsm1–7. Second, we find that deletion of the C‐terminal region of Lsm1 allows Lsm1–7 to bind polyuridine tracts 10‐fold more tightly in the absence of Pat1 or the single adenosine residue. These data support our hypothesis that the C‐terminal region of Lsm1 inhibits association of the ring with RNA, and as such is a negative regulator of RNA binding activity. Finally, we were able to determine a 1.9 Å resolution crystal structure of the S. pombe Lsm1–7 ring (lacking the C‐terminal region of Lsm1) in complex with a polyuridine RNA. This structure shows that the uridine nucleotides bind into the same binding pockets as in Lsm2–8, as hypothesized previously (Montemayor et al., 2018). This work expands our understanding of how the Lsm1–7 complex binds RNA.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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