RNA binding properties of the Lsm1–7 ring from Schizosaccharomyces pombe

Abstract

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.