Variants in LSM7 impair LSM complexes assembly, neurodevelopment in zebrafish and may be associated with an ultra-rare neurological disease.

Leukodystrophies, genetic neurodevelopmental and/or neurodegenerative disorders of cerebral white matter, result from impaired myelin homeostasis and metabolism. Numerous genes have been implicated in these heterogeneous disorders; however, many individuals remain without a molecular diagnosis. Using whole-exome sequencing, biallelic variants in LSM7 were uncovered in two unrelated individuals, one with a leukodystrophy and the other who died in utero. LSM7 is part of the two principle LSM protein complexes in eukaryotes, namely LSM1-7 and LSM2-8. Here, we investigate the molecular and functional outcomes of these LSM7 biallelic variants in vitro and in vivo. Affinity purification-mass spectrometry of the LSM7 variants showed defects in the assembly of both LSM complexes. Lsm7 knockdown in zebrafish led to central nervous system defects, including impaired oligodendrocyte development and motor behavior. Our findings demonstrate that variants in LSM7 cause misassembly of the LSM complexes, impair neurodevelopment of the zebrafish, and may be implicated in human disease. The identification of more affected individuals is needed before the molecular mechanisms of mRNA decay and splicing regulation are added to the categories of biological dysfunctions implicated in leukodystrophies, neurodevelopmental and/or neurodegenerative diseases.

Mutagenic Analysis of the C-Terminal Extension of Lsm1.

The Sm-like proteins (also known as Lsm proteins) are ubiquitous in nature and exist as hexa or heptameric RNA binding complexes. They are characterized by the presence of the Sm-domain. The Lsm1 through Lsm7 proteins are highly conserved in eukaryotes and they form a hetero-octameric complex together with the protein Pat1. The Lsm1-7-Pat1 complex plays a key role in mRNA decapping and 3'-end protection and therefore is required for normal mRNA decay rates in vivo. Lsm1 is a key subunit that is critical for the unique RNA binding properties of this complex. We showed earlier that unlike most Sm-like proteins, Lsm1 uniquely requires both its Sm domain and its C-terminal extension to contribute to the function of the Lsm1-7-Pat1 complex and that the C-terminal segment can associate with the rest of the complex and support the function even in trans. The studies presented here identify a set of residues at the very C-terminal end of Lsm1 to be functionally important and suggest that these residues support the function of the Lsm1-7-Pat1 complex by facilitating RNA binding either directly or indirectly.

The Lsm1-7-Pat1 complex promotes viral RNA translation and replication by differential mechanisms.

The Lsm1-7-Pat1 complex binds to the 3' end of cellular mRNAs and promotes 3' end protection and 5'-3' decay. Interestingly, this complex also specifically binds to cis-acting regulatory sequences of viral positive-strand RNA genomes promoting their translation and subsequent recruitment from translation to replication. Yet, how the Lsm1-7-Pat1 complex regulates these two processes remains elusive. Here, we show that Lsm1-7-Pat1 complex acts differentially in these processes. By using a collection of well-characterized lsm1 mutant alleles and a system that allows the replication of Brome mosaic virus (BMV) in yeast we show that the Lsm1-7-Pat1 complex integrity is essential for both, translation and recruitment. However, the intrinsic RNA-binding ability of the complex is only required for translation. Consistent with an RNA-binding-independent function of the Lsm1-7-Pat1 complex on BMV RNA recruitment, we show that the BMV 1a protein, the sole viral protein required for recruitment, interacts with this complex in an RNA-independent manner. Together, these results support a model wherein Lsm1-7-Pat1 complex binds consecutively to BMV RNA regulatory sequences and the 1a protein to promote viral RNA translation and later recruitment out of the host translation machinery to the viral replication complexes.

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

A major mRNA decay pathway in eukaryotes is initiated by deadenylation followed by decapping of the oligoadenylated mRNAs and subsequent 5'-to-3' exonucleolytic degradation of the capless mRNA. In this pathway, decapping is a rate-limiting step that requires the hetero-octameric Lsm1-7-Pat1 complex to occur at normal rates in vivo. This complex is made up of the seven Sm-like proteins, Lsm1 through Lsm7, and the Pat1 protein. It binds RNA and has a unique binding preference for oligoadenylated RNAs over polyadenylated RNAs. Such binding ability is crucial for its mRNA decay function in vivo. In order to determine the contribution of Pat1 to the function of the Lsm1-7-Pat1 complex, we compared the RNA binding properties of the Lsm1-7 complex purified from pat1Δ cells and purified Pat1 fragments with that of the wild-type Lsm1-7-Pat1 complex. Our studies revealed that both the Lsm1-7 complex and purified Pat1 fragments have very low RNA binding activity and are impaired in the ability to recognize the oligo(A) tail on the RNA. However, reconstitution of the Lsm1-7-Pat1 complex from these components restored these abilities. We also observed that Pat1 directly contacts RNA in the context of the Lsm1-7-Pat1 complex. These studies suggest that the unique RNA binding properties and the mRNA decay function of the Lsm1-7-Pat1 complex involve cooperation of residues from both Pat1 and the Lsm1-7 ring. Finally our studies also revealed that the middle domain of Pat1 is essential for the interaction of Pat1 with the Lsm1-7 complex in vivo.

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

Lsm proteins are a ubiquitous family of proteins characterized by the Sm-domain. They exist as hexa- or heptameric RNA-binding 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 5'-3' mRNA decay pathway. This complex also protects mRNA 3'-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 3'-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 3'-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.

Activation of decapping involves binding of the mRNA and facilitation of the post-binding steps by the Lsm1-7-Pat1 complex.

Decapping is a critical step in the conserved 5'-to-3' mRNA decay pathway of eukaryotes. The hetero-octameric Lsm1-7-Pat1 complex is required for normal rates of decapping in this pathway. This complex also protects the mRNA 3'-ends from trimming in vivo. To elucidate the mechanism of decapping, we analyzed multiple lsm1 mutants, lsm1-6, lsm1-8, lsm1-9, and lsm1-14, all of which are defective in decapping and 3'-end protection but unaffected in Lsm1-7-Pat1 complex integrity. The RNA binding ability of the mutant complex was found to be almost completely lost in the lsm1-8 mutant but only partially impaired in the other mutants. Importantly, overproduction of the Lsm1-9p- or Lsm1-14p-containing (but not Lsm1-8p-containing) mutant complexes in wild-type cells led to a dominant inhibition of mRNA decay. Further, the mRNA 3'-end protection defect of lsm1-9 and lsm1-14 cells, but not the lsm1-8 cells, could be partly suppressed by overproduction of the corresponding mutant complexes in those cells. These results suggest the following: (1) Decapping requires both binding of the Lsm1-7-Pat1 complex to the mRNA and facilitation of the post-binding events, while binding per se is sufficient for 3'-end protection. (2) A major block exists at the post-binding steps in the lsm1-9 and lsm1-14 mutants and at the binding step in the lsm1-8 mutant. Consistent with these ideas, the lsm1-9, 14 allele generated by combining the mutations of lsm1-9 and lsm1-14 alleles had almost fully lost the RNA binding activity of the complex and behaved like the lsm1-8 mutant.

Lsm1-7-Pat1 complex: a link between 3' and 5'-ends in mRNA decay?

Messenger RNA decay occurs via two major pathways (3' to 5' and 5' to 3' pathways) that are conserved in all eukaryotes. A key feature of these pathways is that mRNAs are targeted for degradation only after they have undergone deadenylation so that polyadenylated mRNAs are not attacked. Thus, in the 5' to 3' pathway, oligoadenylated mRNAs (but not polyadenylated mRNAs) are selectively decapped and then degraded in a 5' to 3' exonucleolytic manner. Here, normal rates of decapping require the decapping activator, the Lsm1-7-Pat1 complex. This complex has a strong intrinsic binding preference for oligoadenylated mRNAs over polyadenylated mRNAs and such preferential binding ability is crucial for its mRNA decay function. This suggests that this complex contributes to the deadenylation dependence of decapping in the 5' to 3' pathway by selectively targeting oligoadenylated messages for decapping. The Lsm1-7-Pat1 complex is also capable of recognizing the presence of U-tracts at the 3'-end of RNA and such recognition appears to be important in facilitating the decapping and 5' to 3' decay of histone mRNAs in response to oligouridylation. Thus, this complex influences decapping event at the 5'-end by recognizing simple sequence features at the 3'-end of the mRNA. Additional studies implicate this complex in the inhibition of exosome mediated 3' to 5' decay of mRNAs raising the possibility that in vivo, the major mode of decay of an mRNA could be determined by the efficiency of binding of the Lsm1-7-Pat1 complex to that mRNA.

Roles of eukaryotic Lsm proteins in the regulation of mRNA function.

The eukaryotic Lsm proteins belong to the large family of Sm-like proteins, which includes members from all organisms ranging from archaebacteria to humans. The Sm and Lsm proteins typically exist as hexameric or heptameric complexes in vivo and carry out RNA-related functions. Multiple complexes made up of different combinations of Sm and Lsm proteins are known in eukaryotes and these complexes are involved in a variety of functions such as mRNA decay in the cytoplasm, mRNA and pre-mRNA decay in the nucleus, pre-mRNA splicing, replication dependent histone mRNA 3'-end processing, etc. While most Lsm proteins function in the form of heteromeric complexes that include other Lsm proteins, some Lsm proteins are also known that do not behave in that manner. Abnormal expression of some Lsm proteins has also been implicated in human diseases. The various roles of eukaryotic Lsm complexes impacting mRNA function are discussed in this review.

Purification and analysis of the decapping activator Lsm1p-7p-Pat1p complex from yeast.

Biochemical analysis of the components of the mRNA decay machinery is crucial to understand the mechanisms of mRNA decay. The Lsm1p-7p-Pat1p complex is a key activator of decapping in the 5' to 3'-mRNA decay pathway that is highly conserved in all eukaryotes. The first step in this pathway is poly(A) shortening that is followed by the selective decapping and subsequent 5' to 3'-exonucleolytic degradation of the oligoadenylated mRNAs. Earlier studies suggested that the Lsm1p-7p-Pat1p complex preferentially associates with oligoadenylated mRNAs and facilitates their decapping in vivo (Tharun and Parker, 2001a; Tharun et al., 2000). They also showed that the Lsm1p through Lsm7p and Pat1p are involved in protecting the 3'-ends of mRNAs in vivo from trimming (He and Parker, 2001). Therefore, to gain better insight into the biologic function of the Lsm1p-7p-Pat1p complex, it is important to determine its in vitro RNA binding properties. Here I describe the methods we use in my laboratory for the purification and in vitro RNA binding analysis of this complex from the budding yeast Saccharomyces cerevisiae. Purification was achieved with tandem affinity chromatography using a split-tag strategy. This involved use of a strain expressing FLAG-tagged Lsm1p and 6xHis-tagged Lsm5p and purification by a two-step procedure with an anti-FLAG antibody matrix followed by a Ni-NTA matrix. The purified complex was analyzed for its RNA binding properties with gel mobility shift assays. Such analyses showed that this complex has the intrinsic ability to distinguish between oligoadenylated and polyadenylated RNAs and that it binds near the 3'-ends of RNAs (Chowdhury et al., 2007). These observations, therefore, highlighted the importance of the intrinsic RNA binding properties of this complex as key determinants of its in vivo functions.

lsm1 mutations impairing the ability of the Lsm1p-7p-Pat1p complex to preferentially bind to oligoadenylated RNA affect mRNA decay in vivo.

The poly(A) tail is a crucial determinant in the control of both mRNA translation and decay. Poly(A) tail length dictates the triggering of the degradation of the message body in the major 5' to 3' and 3' to 5' mRNA decay pathways of eukaryotes. In the 5' to 3' pathway oligoadenylated but not polyadenylated mRNAs are selectively decapped in vivo, allowing their subsequent degradation by 5' to 3' exonucleolysis. The conserved Lsm1p-7p-Pat1p complex is required for normal rates of decapping in vivo, and the purified complex exhibits strong binding preference for oligoadenylated RNAs over polyadenylated or unadenylated RNAs in vitro. In the present study, we show that two lsm1 mutants produce mutant complexes that fail to exhibit such higher affinity for oligoadenylated RNA in vitro. Interestingly, these mutant complexes are normal with regard to their integrity and retain the characteristic RNA binding properties of the wild-type complex, namely, binding near the 3'-end of the RNA, having higher affinity for unadenylated RNAs that carry U-tracts near the 3'-end over those that do not and exhibiting similar affinities for unadenylated and polyadenylated RNAs. Yet, these lsm1 mutants exhibit a strong mRNA decay defect in vivo. These results underscore the importance of Lsm1p-7p-Pat1p complex-mRNA interaction for mRNA decay in vivo and imply that the oligo(A) tail mediated enhancement of such interaction is crucial in that process.

The decapping activator Lsm1p-7p-Pat1p complex has the intrinsic ability to distinguish between oligoadenylated and polyadenylated RNAs.

Decapping is a critical step in mRNA decay. In the 5'-to-3' mRNA decay pathway conserved in all eukaryotes, decay is initiated by poly(A) shortening, and oligoadenylated mRNAs (but not polyadenylated mRNAs) are selectively decapped allowing their subsequent degradation by 5' to 3' exonucleolysis. The highly conserved heptameric Lsm1p-7p complex (made up of the seven Sm-like proteins, Lsm1p-Lsm7p) and its interacting partner Pat1p activate decapping by an unknown mechanism and localize with other decapping factors to the P-bodies in the cytoplasm. The Lsm1p-7p-Pat1p complex also protects the 3'-ends of mRNAs in vivo from trimming, presumably by binding to the 3'-ends. In order to determine the intrinsic RNA-binding properties of this complex, we have purified it from yeast and carried out in vitro analyses. Our studies revealed that it directly binds RNA at/near the 3'-end. Importantly, it possesses the intrinsic ability to distinguish between oligoadenylated and polyadenylated RNAs such that the former are bound with much higher affinity than the latter. These results indicate that the intrinsic RNA-binding characteristics of this complex form a critical determinant of its in vivo interactions and functions.

Mutations in the Saccharomyces cerevisiae LSM1 gene that affect mRNA decapping and 3' end protection.

The decapping of eukaryotic mRNAs is a key step in their degradation. The heteroheptameric Lsm1p-7p complex is a general activator of decapping and also functions in protecting the 3' ends of deadenylated mRNAs from a 3'-trimming reaction. Lsm1p is the unique member of the Lsm1p-7p complex, distinguishing that complex from the functionally different Lsm2p-8p complex. To understand the function of Lsm1p, we constructed a series of deletion and point mutations of the LSM1 gene and examined their effects on phenotype. These studies revealed the following: (i) Mutations affecting the predicted RNA-binding and inter-subunit interaction residues of Lsm1p led to impairment of mRNA decay, suggesting that the integrity of the Lsm1p-7p complex and the ability of the Lsm1p-7p complex to interact with mRNA are important for mRNA decay function; (ii) mutations affecting the predicted RNA contact residues did not affect the localization of the Lsm1p-7p complex to the P-bodies; (iii) mRNA 3'-end protection could be indicative of the binding of the Lsm1p-7p complex to the mRNA prior to activation of decapping, since all the mutants defective in mRNA 3' end protection were also blocked in mRNA decay; and (iv) in addition to the Sm domain, the C-terminal domain of Lsm1p is also important for mRNA decay function.