Both Sm-domain and C-terminal extension of Lsm1 are important for the RNA-binding activity of the Lsm1–7–Pat1 complex

Abstract

Lsm proteins are a ubiquitous family of proteins characterized by the Sm-domain. They exist as hexa-or heptameric RNAbinding complexes and carry out RNA-related functions. The Sm-domain is thought to be sufficient for the RNA-binding activity of these proteins. The highly conserved eukaryotic Lsm1 through Lsm7 proteins are part of the cytoplasmic Lsm1-7-Pat1 complex, which is an activator of decapping in the conserved 59-39 mRNA decay pathway. This complex also protects mRNA 39-ends from trimming in vivo. Purified Lsm1-7-Pat1 complex is able to bind RNA in vitro and exhibits a unique binding preference for oligoadenylated RNA (over polyadenylated and unadenylated RNA). Lsm1 is a key subunit that determines the RNA-binding properties of this complex. The normal RNA-binding activity of this complex is crucial for mRNA decay and 39-end protection in vivo and requires the intact Sm-domain of Lsm1. Here, we show that though necessary, the Sm-domain of Lsm1 is not sufficient for the normal RNA-binding ability of the Lsm1-7-Pat1 complex. Deletion of the C-terminal domain (CTD) of Lsm1 (while keeping the Sm-domain intact) impairs mRNA decay in vivo and results in Lsm1-7-Pat1 complexes that are severely impaired in RNA binding in vitro. Interestingly, the mRNA decay and 39-end protection defects of such CTD-truncated lsm1 mutants could be suppressed in trans by overexpression of the CTD polypeptide. Thus, unlike most Sm-like proteins, Lsm1 uniquely requires both its Sm-domain and CTD for its normal RNA-binding function.

Pat1 contributes to the RNA binding activity of the Lsm1-7–Pat1 complex

“…This may partly be to do with the smaller size of the Lsm1-7 complex compared with the wild-type Lsm1-7-Pat1 complex (∼97 kD vs. ∼185 kD). Second, while the Lsm1-7-Pat1 complex clearly exhibited a higher affinity for the PGK1-A 5 RNA than the PGK1 RNA as expected and as observed earlier (Chowdhury and Tharun 2009;Chowdhury et al 2012), such binding preference could not be observed with the Lsm1-7 complex. Very similar results were also observed when the experiments were carried out using the MFA2 and MFA2-A 5 RNAs (42-mer RNA derived from the 3 ′ UTR of MFA2 and the 3 ′ -penta-adenylated version of such RNA) as substrates for gel shift assays (Fig.…”

“…The ability of the Lsm1-7-Pat1 complex to bind RNA and to recognize the presence of the 3 ′ oligo(A) tail of the RNA is essential for mRNA decay in vivo as revealed by our past analysis of various lsm1 mutants (Tharun et al 2005;Tharun 2008, 2009;Chowdhury et al 2012). However, the contribution of Pat1 to the RNA binding properties of the Lsm1-7-Pat1 complex is not known.…”

“…Our earlier studies support the idea that the lack of binding preference for oligoadenylated RNA of the Lsm1-7 complex and the Pat1 fragments observed here is not because the unadenylated RNA binding activities measured are very low. For example, although the mutant complexes isolated from the lsm1-27 and lsm1-28 strains (that carry large deletions in the C-terminal domain of Lsm1) are severely impaired in binding unadenylated RNA, their binding preference for oligoadenylated RNA over unadenylated RNA can be very clearly observed (Chowdhury et al 2012). Further, very low binding activity is also observed with the wild-type complex when a poor unadenylated RNA substrate like RPP2B, TEF1(s) or MFA26xUΔ RNA is used (Chowdhury et al 2007).…”

“…The poor binding activity of the Lsm1-7 ring and Pat1 fragments could be simply either because the fewer RNA contacts (compared with the Lsm1-7-Pat1 complex) made by their individual RNA binding surfaces are not sufficient to support significantly stable RNA binding or because their binding surfaces attain optimal conformation only when they associate to form the entire Lsm1-7-Pat1 complex. Our past studies have shown that both the Sm domain and the C-terminal domain of Lsm1 are essential for the normal RNA binding activity of the Lsm1-7-Pat1 complex, suggesting that residues from both of these regions of Lsm1 contribute to the RNA binding surface of the Lsm1-7 ring Tharun 2008, 2009;Chowdhury et al 2012). The other Lsm subunits (Lsm2 through Lsm7) also contact RNA in the Lsm1-7-Pat1 complex, although the manner in which they affect the overall binding is not clear.…”

“…First, although the expected size of yeast Pat1 is 88 kD, it is known to run in SDS-PAGE as multiple bands of anomalous electrophoretic mobility, including two or three bands with mobility ranging from ∼62 kD to ∼75 kD and a ∼97-kD band (Bouveret et al 2000;Chowdhury et al 2007Chowdhury et al , 2012Tharun 2008, 2009). In our purified Lsm1-7-Pat1 complex preparations, we always find the ∼62-kD to ∼75-kD Pat1 bands to be much stronger than the ∼97-kD band (Chowdhury et al 2007(Chowdhury et al , 2012Tharun 2008, 2009). Thus, the ∼65-kD and ∼97-kD bands (wherein the latter band is weaker than the former) observed in UV crosslinking match the typical mobility of yeast Pat1 bands.…”