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Originally published In Press as doi:10.1074/jbc.M400678200 on February 26, 2004

J. Biol. Chem., Vol. 279, Issue 18, 18210-18219, April 30, 2004
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Identification and Functional Characterization of Lsm Proteins in Trypanosoma brucei*

Qing Liu{ddagger}, Xue-hai Liang{ddagger}, Shai Uliel, Myriam Belahcen, Ron Unger, and Shulamit Michaeli§

From the Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel

Received for publication, January 21, 2004 , and in revised form, February 23, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
RNA interference of Sm proteins in Trypanosoma brucei demonstrated that the stability of the small nuclear RNAs (U1, U2, U4, U5) and the spliced leader RNA, but not U6 RNA, were affected upon Sm depletion (Mandelboim, M., Barth, S., Biton, M., Liang, X. H., and Michaeli, S. (2003) J. Biol. Chem. 278, 51469–51478), suggesting that Lsm proteins that bind and stabilize U6 RNA in other eukaryotes should exist in trypanosomes. In this study, we identified seven Lsm proteins (Lsm2p to Lsm8p) and examined the function of Lsm3p and Lsm8p by RNA interference silencing. Both Lsm proteins were found to be essential for U6 stability and mRNA decay. Silencing was lethal, and cis- and trans-splicing were inhibited. Importantly, silencing also affected the level of U4.U6 and the U4.U6/U5 tri-small nuclear ribonucleoprotein complexes. The presence of Lsm proteins in trypanosomes that diverged early in the eukaryotic lineage suggests that these proteins are highly conserved in both structure and function among eukaryotes. Interestingly, however, Lsm1p that is specific to the mRNA decay complex was not identified in the genome data base of any kinetoplastidae, and the Lsm8p that in other eukaryotes exclusively functions in U6 stability was found to function in trypanosomes also in mRNA decay. These data therefore suggest that in trypanosomes only a single Lsm complex may exist.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Pre-mRNA splicing requires five small nuclear RNPs1: the U1, U2, U4, U5, and U6 snRNPs. The first four snRNPs consist of an RNA molecule and core proteins, known as Sm proteins, as well as particle-specific proteins. In vertebrates, the binding of Sm proteins to the snRNA takes place in the cytoplasm and is required for the hypermethylation of the 5' cap structure and for the import of snRNPs into the nucleus (1). The Sm proteins are low molecular mass proteins ranging in size from 8 to 28 kDa and are characterized by bipartite sequence motifs, termed Sm 1 and Sm 2 (2, 3). The Sm motifs function in protein-protein interactions (4) and are separated by a variable loop sequence. The seven Sm proteins, SmB, D1, D2, D3, E, F, and G, assemble to snRNP from B.D3, D1.D2, and E.F.G sub-particles to form a doughnut-shaped complex (5). None of the individual Sm proteins binds stably to RNA; instead, the Sm proteins interact via their Sm motifs to form a heptameric ring around the snRNAs. The RNA binding site is generated only after the formation of Sm protein heteromers (6).

In addition to the canonical Sm proteins, other proteins carrying the Sm motifs have been identified in many eukaryotes. In yeast, nine such proteins exist that were designated Lsm (Sm-like) proteins. Two functionally distinct heptameric complexes of these Lsm proteins were characterized. One complex containing Lsm2p-Lsm8p binds to the 3' end U tract of U6 snRNA in the nucleus and is required for the stable accumulation of U6 snRNA (7, 8). In addition, these proteins are required for the formation of the U4.U6 and U4.U6/U5 complexes (8, 9). Depletion of Lsm proteins caused splicing defects, resulting in the accumulation of pre-mRNA (9). Interestingly, the Lsm2p-Lsm8p complex purified from human cells was found to form a doughnut-shaped structure analogous to the structure formed in the canonical Sm complex, except that the Lsm complex formation can occur in the absence of RNA (8). Among these Lsm proteins, five (Lsm2p-5p and Lsm8p) are essential for cell growth (10).

The second Lsm complex, consisting of Lsm1p-Lsm7p, functions in mRNA decay in the cytoplasm, most probably in the decapping step. By tagging the Lsm1p protein in yeast, it was demonstrated that the complex also carries, in addition to the Lsm1p-7p proteins, the Pat1 and Xrn1 exonucleases (11). It was also shown that the yeast Lsm1p mutant stabilized mRNAs (12) and that depletion of Lsm1p, 6p, and 7p in yeast affected mRNA stability, suggesting that the Lsm1p-7p complex is required for mRNA decay (11). The function and components of these complexes seem to be also conserved in humans, because the Lsm1p homologue, CaSm, was not found in human U4.U6/U5 complex (8), and Lsm8p was not co-localized with mRNA degradation enzymes (Dcp1/2 and Xrn1) in cytoplasm (13).

Lsm proteins appear to have additional functions apart from stabilization of U6 snRNA and mRNA. The Lsm2p-8p complex was also shown to bind U8 snoRNA in Xenopus (14), and yeast U7 snoRNA binds to Lsm proteins (15), suggesting that Lsm proteins may play a role in the stabilization of other small stable RNAs. In yeast, Lsm proteins were shown to be required for pre-tRNA and pre-rRNA processing (10, 16). Lsm2p-7p but not other Lsm proteins associate with pre-RNase P RNA (17). The existence of a Sm-like protein SmAP in Archaea implies that Sm proteins had an ancient origin in nature (18). In fact, an Sm motif containing protein, Hfq, was also found in Escherichia coli (19). These proteins are active only after the formation of a ring structure (20, 21). Hfq protein was shown to interact with many different regulatory RNAs and to affect the translation and stability of several mRNAs (19). In addition, Hfq also mediates RNA-RNA interactions (22).

Trypanosomes are protozoan parasites that diverged very early in the eukaryotic lineage (23). These organisms participate in unique RNA processing events such as trans-splicing and RNA editing (24, 25). Trans-splicing evolved to separate long polycistronic transcripts. In trypanosomes, all mRNAs undergo trans-splicing, but cis-splicing also exists (26). All spliceosomal U snRNAs were identified in this organism (24). As opposed to their eukaryotic counterparts, the trypanosome U snRNAs are transcribed by RNA polymerase III (27). Trypanosome SL RNA, U1, U2, U4, and U5 RNAs associate with Smcore proteins (28–31). The Sm binding sites on the snRNAs were found to be divergent compared with the canonical site (32). The first Sm protein was identified as an SL RNP core protein in Leptomonas collosoma (29). Later, the full set of seven Sm proteins was identified in Trypanosoma brucei and Trypanosoma cruzi (30), and the proteins have the potential to form the canonical heptameric ring-like structure. The Sm proteins carry bipartite Sm motifs including interesting deviations from the Sm consensus (30). In addition, the SmD1 and SmD3 lack the C-terminal RG dipeptide repeats of the human homologues (30). The arginine in this domain is methylated in the mammalian protein, constituting an important determinant of the Sm epitope (33). The lack of these modifications on the trypanosome proteins may partly explain why the trypanosome Sm proteins are not recognized by the Sm antibodies (28). Recently, we silenced two Sm proteins by RNAi in T. brucei. Interestingly, whereas the level of U1, U2, U4, and U5 RNAs was reduced, no changes were observed on the level of U6 and the trypanosome-specific small RNA (SLA1) that was shown to guide pseudouridylation on the SL RNA (34). These data suggest that neither U6 nor SLA1 binds to the canonical Sm proteins.

In this study, seven Lsm proteins homologous to the complex Lsm2p-8p were identified in the T. brucei genome data base. The function of two proteins, Lsm3p and Lsm8p, was examined by RNAi silencing. The results indicate that these Lsm proteins associate with U6 snRNA and are required for both trans- and cis-splicing. In the Lsm8-silenced cell line, the level of the U4/U6 and U4.U6/U5 complexes was reduced. Interestingly, Lsm8p that exclusively function in U6 stability in other eukaryotes was found to function in trypanosomes also in mRNA decay. These results suggest that in trypanosomes that diverged early in evolution of eukaryotes the Lsm proteins are highly conserved in both structure and function. However, this organism may possess a single complex that functions in both U6 RNP biogenesis and mRNA decay.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Constructs and Establishment of Stable Cell Lines—The stem-loop construct for silencing Lsm8 was established as described in (35). Briefly, a 400-bp coding region sequence derived from the Lsm8 (GenBankTM accession number AY551266 [GenBank] ) gene obtained from the genome data base was amplified with oligonucleotides L8001 and L8003, containing the XbaI and HindIII sites, respectively, and subcloned into the NheI and HindIII site of the pJM326 vector. The PCR fragment was fused with the Pex 11 sequence present in the vector and the 950-bp fused fragment was released from the XbaI and HindIII sites. The same region was PCR-amplified with primers L8001 and L8004, containing the XbaI and MluI sites, respectively. This fragment was subcloned into the Plew 100 vector. The plasmid was further linearized with XbaI and HindIII and ligated with the 950-bp fused fragment derived from pJM326 vector. To silence the Lsm3 gene expression, a 300-bp coding sequence (GenBankTM accession number AY551261 [GenBank] ) was amplified from the T. brucei genome with oligonucleotides L3001 and L3002, and the PCR fragment was cloned into the XhoI and HindIII sites of the pZJM vector, in between the two opposing T7 promoters. The constructs expressing dsRNA were transfected into the 29–13 parental cell line, and the transformants were selected with 5 µg/ml phelomycin and cloned by limited dilution. The expression of dsRNA was triggered by the addition of 5 µg/ml tetracycline.

Oligonucleotides—Oligonucleotides used were as follows: L8001, 5'-GCTCTAGAATGCTTTCCCAATATCTCAGG-3', sense, specific to the Lsm8 gene, from position 1 to 20 relative to the start codon, containing an XbaI site; L8003, 5'-CCCAAGCTTCTATCCACCTTTAACTGAA-3', antisense, complementary to the Lsm8 gene, from position 371 to 389, containing a HindIII site; L8004, 5'-CGACGCGTCTATCCACCTTTAACTGAA-3', antisense, complementary to the Lsm8 gene, from position 371 to 389, containing a MluI site; L8002, 5'-GCCTCCTCCACCGTATTA-3', antisense, complementary to the Lsm8 gene, from position 282 to 299; L3001, 5'-CCGCTCGAGATGCAAACTTCTCGCGCC-3', sense, specific to Lsm3, from –13 to –30, relative to the start codon, containing a XhoI site; L3002, 5'-CCCAAGCTTAACCTGATACGATTGGGG-3', antisense, complementary to Lsm3, from 223 to 240, relative to the start codon, containing a HindIII site; 45338, 5'-CGCTATTATTAGAACAGTTTCTG-3', sense, specific to SL RNA, from position 7 to 29; Tubulin-3A, 5'-ATGCAGATAGCCTCACGC-3', antisense, complementary to the {alpha}-tubulin gene, from position 3 to 20, relative to the start codon; SLA-S, 5'-AAAGCTCTTTTATGTAGTGTG-3', sense, specific to SLA1 RNA, from position 1 to 21; 76A,5'-TCAGTCTGGGCACAATT-3', antisense, complementary to snoRNA-76 in the SLA1 locus, from position 54 to 70; 92A, 5'-TCAACGTCCATCTGCGACGG-3', antisense, complementary to snoRNA-92 in the SLA1 locus, from position 31 to 50; TBU1–3A, 5'-CGCTTTCGTTCCCAC-3', antisense, complementary to U1 snRNA, from position 54 to 68; 14824, 5'-AGCTAAAAAGCCGAGAAGATAT-3', antisense, complementary to U2 snRNA, from position 34 to 45; 31253, 5'-TCTYGCTCTCCAGTTTCRTG-3', antisense, complementary to SLA1 RNA, from position 51 to 70; 12407, 5'-AGCTATATCTCTCGAA-3', antisense, complementary to U6 snRNA, from position 81 to 95; 17036, 5'-GTACCGGATATAGT-3', antisense, complementary to U4 snRNA, from 60 to 75; 16513, 5'-CCGCTCGAGGACACCCCAAAGTTT-3', antisense, complementary to U5 snRNA, from position 48 to 62; 76S, 5'-AACATCACAGACTTTGA-3', sense, specific to snoRNA-76 in the SLA locus, from position 18 to 34; TB7SL-5A, 5'-GAACCCCCGCTTGTCC-3', antisense, complementary to 7SL RNA, from 60 to 75; P0001, 5'-CCCAACGAAACCACTAGAGG-3', sense, specific to the first exon of PAP gene, from 223 to 242, relative to the start codon; P0004, 5'-CTGTTCGTCGTCGTGGCA-3', sense, specific to PAP cis-spliced intron, upstream from 3' SS, from –133 to –150; P0003, 5'-ATCGACAGCTGTGCCTCTG-3', antisense, complementary to PAP gene, located 136 bp downstream from the 3' SS; 9091, 5'-TGCGTCTGTTGGCCC-3', antisense, complementary to SL RNA, from position 65 to 79; 19796, 5'-CGCCGTTTGCGTTCA-3', antisense, complementary to 5.8S rRNA, from position 101 to 115; Sno1–5XH, 5'-CCGCTCGAGTTACCTATTCCATGCACATT-3', sense, carrying a XhoI site, specific to the upstream flanking sequence of T. brucei TBC-4 snoRNA, from position –44 to –63; Sno1–3H, 5'-CCCAAGCTTGAGAAGAGTGTGCACTAACC-3', antisense, carrying a HindIII site, complementary to the downstream flanking sequence of T. brucei TBH1 snoRNA, from position +16 to +35; and 44252, 5'-GGGTGAACAATCCAACCCTT-3', antisense, complementary to 28 S rRNA, from position 3673 to 3692 (T. brucei, GenBankTM accession number X14553 [GenBank] ).

Northern Analysis—Total RNA (~30 µg/lane) from induced and uninduced cells was separated on 1.2% agarose-formaldehyde gel and transferred onto the membrane. The blot was hybridized at 42 °C overnight with a randomly labeled DNA fragment specific to Lsm3 mRNA or with an end-labeled oligonucleotide complementary to {alpha}-tubulin. The same membrane was hybridized again with a 5' end-labeled oligonucleotide complementary to 7SL RNA, to examine the amount of RNA in each lane. After hybridization, the membrane was washed at 55 °C when DNA probes were used or at 42 °C when oligonucleotide probes were used. Washing was performed twice, 20 min each with 2x SSC in the presence of 0.1% SDS. To detect the snRNAs, RNA was prepared from sucrose gradient fractions and separated on 10% polyacrylamide gel. The membrane was hybridized at 42 °C with 5' end-labeled oligonucleotides complementary to U4, U5, and U6 RNA, respectively.

Primer Extension Analysis—Total RNA (10 µg) from induced and uninduced cells was subjected to primer extension analysis as described (36). 5' end-labeled oligonucleotides were used in the extensions to detect the level of U1, U2, U4, U5, U6, SLA1, and SL RNA. To determine the amount of RNA, the level of snoRNA-92 was examined. The extension products were separated on 6% polyacrylamide denaturing gel.

RT-PCR—Total RNA (50 µg) was incubated with 50 units of RNase-free DNase (RQ I; Promega) at 37 °C for 1 h. After phenol:chloroform extraction, 5 µg of RNA (DNA-free) was used in reverse transcription with 1 unit of reverse transcriptase (RT; Roche Applied Science) in the presence of 100 pmol random primer (Promega). The reverse transcription was performed at 42 °C for 1 h. The cDNA was used in subsequent PCR reactions.

To detect the level of Lsm8 mRNA, oligonucleotides 45338 and L8002 were used to detect the mature mRNA. Oligonucleotides SLA-S and TB-76 were used to detect the level of SLA1 precursor that serves as a control for the quantity and quality of cDNA. To detect the level of mature PAP mRNA, oligonucleotides P0001 and P0003 were used. P0004 and P0003 were used to detect the level of pre-mRNA in PAP.To examine the amount of template used in the PCR reaction, oligonucleotides 76S and 92a were used in the analysis of Lsm8 silencing to amplify the snoRNA precursor in the SLA1 locus or Sno1–5XH and Sno1–3H in the analysis of Lsm3 silencing to detect the TBh1 snoRNA locus. The PCR reaction was performed under the following conditions: 94 °C, 30 s; 60 °C, 1 min; 72 °C, 1 min; 30 cycles.

To detect the DNA contamination, DNA-removed total RNA was directly used in a PCR reaction with oligonucleotides SLA-S and Tb-76 to amplify the SLA1 locus in the analysis of Lsm8 mRNA (see Fig. 2) or 92a and 76S in the PAP analysis (see Fig. 6). The RT-PCR products were analyzed on 2% agarose gel and visualized by EtBr staining.



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  		FIG. 2.
Lsm8p and Lsm3p are required for cell growth. A, silencing of Lsm8 by RNAi. Panel a, schematic representation of Lsm8 mRNA detection by RT-PCR reaction; panel b, RT-PCR analysis. The RT-PCR reaction was separated on 2% agarose gel. Templates used in lanes 1, 3, and 5 were RNA from uninduced cells. RNA from silenced cells was used in lanes 2, 4, and 6. Lanes 1 and 2, RT-PCR was performed using primers 45338 and L8002 to detect the spliced mRNA. Lanes 3 and 4, PCR was performed on RNA using primers SLA-S and Tb-76 (to control DNA contamination). Lanes 5 and 6, RT-PCR was performed on cDNA using primers SLA-S and Tb-76 to control the quantity and quality of the cDNA. RT-PCR reaction was performed as described under "Experimental Procedures." Panel c, growth of Lsm8-silenced cells upon tetracycline induction. B, silencing of Lsm3. Total RNA (20 µg) from induced (+Tet) and uninduced cells (–Tet) was separated on 1.2% agarose gel and subjected to Northern analysis. The membrane was hybridized with a DNA probe specific to Lsm3 mRNA and 5.8 S rRNA; panel b, cell growth upon tetracycline induction.

 



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  		FIG. 6.
Cis-splicing of PAP was affected upon the silencing of both Lsm8 and Lsm3. A, schematic representation of the RT-PCR reaction. B, RT-PCR to detect the level of PAP transcript upon the silencing of Lsm8. RT-PCR was performed as described under "Experimental Procedures." The PCR reaction was analyzed on 2% agarose gel and visualized by EtBr staining. The mature mRNA is designated by a white arrow, and the precursor is indicated by an open arrow. The precursor of SLA1 locus is marked with a sharp arrow. Templates used in the reactions were as follows: lanes 1, 3, and 5, cDNA from unsilenced cells; lanes 2, 4, and 6, cDNA from induced cells; lanes 7 and 8, RNA from uninduced and induced cells, respectively (control for DNA contamination). Primers used were as follows: lanes 1 and 2, primers for the mature PAP mRNA; lanes 3 and 4, primers for the precursor of PAP; lanes 5–8, primers for the precursor of SLA1 locus (control for the quantity and quality of the cDNA). M, the 1-kb ladder. C, silencing of Lsm3. RT-PCR was performed as described in B. Templates used in the reactions were as follows: lanes 1, 3, and 7, cDNA from uninduced cells; lanes 2, 4, and 8, cDNA from induced cells; and lanes 5 and 6, RNA from uninduced and induced cells, respectively. Primers used were as follows: lanes 1 and 2, primers for the mature PAP mRNA; lanes 3 and 4, primers for the precursor of PAP; lanes 5–8, primers to detect the precursor from the snoRNA TBH1 locus (control for the quality and quantity of cDNA). The products were analyzed on 2% agarose gel.

 
Sucrose Gradient Fractionation—Lsm8 silenced or unsilenced cells (109 log-phase) were harvested and washed with 1x phosphate-buffered saline and Buffer A (35 mM HEPES (pH 7.9), 10 mM MgCl2, 24 mM KCl, 5 mM {beta}-mercaptoethanol). Cell pellet was resuspended in 600 µl of Buffer A, and Nonidet P-40 was added to make a 0.1% final concentration. Cells were broken by pouncing 20 times with a type A pestle. Salt extracts were prepared by the stepwise addition of KCl to 200 mM. The cell lysate was cleared by 20 min of centrifugation at 10,000 x g. The whole cell extract was layered on 10–30% sucrose gradient containing 200 mM KCl. The gradient was fractionated by centrifugation at 35,000 rpm for 22 h using an SW41 rotor. Aliquots were collected from top to bottom, and RNA was prepared from fractions and subjected to Northern analysis.

Measuring the Half-life of mRNA during Lsm Silencing—About 2 x 108 log phase of Lsm8 and Lsm3 silenced cells (3 days after adding tetracycline) or unsilenced T. brucei cells were harvested. The cell pellet was resuspended with fresh medium carrying all antibiotics required for dsRNA expression. The cells were separated into five tubes, 5 ml in each. Next, actinomycin D was added to make a 10 µg/ml final concentration, and the cells were incubated for 0 to 2 h at 27 °C. RNA was prepared and subjected to Northern analysis as described above. The blot was hybridized with randomly labeled probes specific to SRP19, RHS1 mRNA (kindly provided by Elisabetta Ullu) and subsequently with 5' end-labeled oligonucleotide complementary to 28 S rRNA to control the amount of RNA on the blot.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Lsm Proteins in Trypanosoma brucei—Silencing of the Sm proteins showed no effect on the level of U6 snRNA (31), suggesting that Lsm proteins should exist in T. brucei. To identify such proteins, we screened the T. brucei genome data base for Sm motif-containing proteins. In addition to the seven canonical Sm proteins (30), several additional proteins were revealed that carry the conserved Sm motifs. Seven of these proteins were identified as Lsm proteins. The results, presented in Fig. 1, align these proteins with their eukaryotic homologues. The T. brucei Lsm2p-8p proteins share 48.4, 40, 42.5, 30, 35, 56.5, and 42% similarities with their yeast homologues, respectively. Alignment of the Sm1 and Sm2 motifs of these proteins is presented in Fig. 1B, and the consensus profile derived from these sequences is depicted in panel C. Using this profile, we searched different data bases and were able to selectively identify Lsm but not Sm proteins. Interestingly, there are unique features of this profile when it is compared with the profile recently published for the Sm1 motif of T. brucei Sm proteins (panel C) (30). The Lsm Sm motif 1 starts with the sequence VXV; it is longer in the N terminus but shorter at the C terminus; lacking the sequence E, it always carries a leucine at position 15 and an asparagine at position 23 and not MN as in the Sm proteins (Fig. 1C).



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  		FIG. 1.
Identification of Lsm proteins. A, sequence alignment of Lsm proteins. The alignment was performed using the program PILEUP and SeqVu. The Sm motif 1 is denoted by a thick line, and the Sm motif 2 is denoted by a thin line. The yeast Saccharomyces cerevisiae (y) Lsm2p and 4p-7p GenBankTM accession numbers are P38203 [GenBank] , P40070 [GenBank] , P40089 [GenBank] , Q06406 [GenBank] , and NP_014252 [GenBank] , respectively. The human (h) GenBankTM accession numbers of Lsm2p and 4p-7p are Q9Y333, AAH00387 [GenBank] NP_036454 [GenBank] , Q9Y4Y8, and NP_057283 [GenBank] , respectively. The GenBankTM accession numbers forthe T. brucei Lsm2p and Lsm4p-7p were AY551260 [GenBank] , AY551262 [GenBank] , AY551263 [GenBank] , AY551264 [GenBank] , and AY551265 [GenBank] , respectively. The GenBankTM accession numbers of Lsm3p proteins are Caenorhabditis elegans, NP_502579 [GenBank] ; Homo sapiens, NP_057284 [GenBank] ; Arabidopsis thaliana, NP_177812 [GenBank] ; Drosophila melanogaster, NP_732931 [GenBank] ; S. cerevisiae, P57743 [GenBank] ; T. brucei, AY551261 [GenBank] ; for T. cruzi, they are TCGSM95 and TCGPH15. The GenBankTM accession numbers of Lsm8p are H. sapiens, NP_057284 [GenBank] ; D. melanogaster, NP_647660 [GenBank] ; C. elegans, AAL00875 [GenBank] Schizosaccharomyces pombe, NP_588509 [GenBank] ; T. brucei, AY551266 [GenBank] ; T. cruzi, TCGOA79; and Leishmania major, LM25.1.Contig1 (TIGR). The identical amino acids are boxed, and the similar residues are shadowed. B, sequence comparison of Sm motifs in trypanosome Lsm proteins. The conserved residues are in bold, and the conserved region is underlined. C, profiles of trypanosome Lsm and Sm proteins. X designates any amino acid. The conserved residues are in bold.

 
Although trypanosome Lsm proteins carry the conserved Sm motifs, they possess unique features compared with their counterparts in other organisms. Lsm2p (the homologue to SmD1) is longer at both the N and C termini. The C terminus carries multiple arginines. Interestingly, the SmD1 of T. brucei lacks the RG repeats present in the mammalian homologue. This arginine carries the dimethyl modification, which constitutes an important determinant of the Sm epitope. The Lsm3p is shorter at the N terminus. Also, Lsm6p (SmF homologue) is shorter at both the C and N termini. Several differences exist for Lsm8p. First, the gap between the two Sm motifs is much longer in the trypanosome protein. The sequence in this loop is not conserved among the different trypanosome species. Second, most strikingly, an EST exists for this gene (T. brucei EST accession number gb T26050 [GenBank] .1) that lacks the extended sequence between the two motifs, suggesting that this long loop may be removed by splicing. Surprisingly, the intron removed from this cDNA is not flanked by canonical cis-splicing signals (see "Discussion").

Despite these interesting changes, the trypanosome Lsm proteins carry characteristic features specific to individual Lsm proteins. For example, the N terminus of Lsm4p is rich in hydrophobic amino acids, whereas the C terminus is more hydrophilic. This feature also exists in trypanosome Lsm4p protein. In yeast and humans, the gap between the two Sm motifs in Lsm5p and Lsm6p is composed of hydrophilic sequences. The trypanosome counterparts also have such characteristics. This information suggests that the trypanosome Lsm proteins do contain the conserved Lsm motifs and domain features that specify the different Lsm proteins in other organisms.

Lsm3 and Lsm8 Are Essential for Cell Growth—To characterize the role of Lsm proteins, we chose Lsm3p and Lsm8p for functional analysis. We were especially interested in examining the function of Lsm8p, which possesses interesting characteristics described previously. The expression of Lsm proteins was silenced by the two approaches. The Lsm8 was silenced by producing dsRNA in the form of a stem-loop structure, as described under "Experimental Procedures" (35, 37). For silencing the Lsm3 gene, PCR was used to amplify the gene that was cloned into the pZJM vector carrying the two T7 opposing promoters (35). The constructs were linearized to integrate into the non-transcribed rRNA spacer region (35). One day after transfection, the cells were diluted onto the microtiter plates to obtain a clonal population. After 3 weeks the cloned cells were used to establish a pure culture that was examined for inhibition of growth upon the addition of tetracycline. The results, presented in Fig. 2, A, c and B, b, suggest that the two genes are essential for cell growth, because cells stopped growing 4 days after adding tetracycline and eventually died.

To correlate the growth arrest with the silencing of these genes, we examined the level of the Lsm8 and Lsm3 mRNAs by RT-PCR and Northern analysis, respectively, as presented in Fig. 2, A, b and B, a. Upon tetracycline addition, the mature Lsm8 mRNA was almost undetectable; however, a nonspecific product (Fig. 2A, b, marked by an asterisk) was not affected. These results indicate a reduction of more than 90% in the level of these mRNAs. The reduction of the mRNA was the result of dsRNA production. Silencing using the stem-loop was, however, more stable than that using opposing T7 promoters. Silencing by the stem-loop system was not lost even after prolonged propagation.

Lsm3p and Lsm8p Are Required for the Accumulation of U6 but Not Other U snRNAs—To examine whether Lsm3p and Lsm8p function in U6 stability, like their mammalian counterparts (9), we examined the level of snRNAs during Lsm3 and Lsm8 silencing. RNA was prepared from Lsm8 silenced and unsilenced cells and subjected to primer extension analysis. As shown in Fig. 3A, U1, U2, U4, and U5 were not affected upon silencing; however, the level of U6 RNA was reduced by 87%. The level of U3 snoRNA was used to control the amount of RNA in each sample. Similar results were obtained for Lsm3 silencing (Fig. 3B). These results suggest that the two Lsm proteins are required for U6 stability. No effect on the level of SLA1 was observed during the silencing of these genes. These data, together with the results obtained during Sm silencing (31), suggest that neither Sm nor Lsm proteins bind to SLA1. Indeed, we have demonstrated recently that the level of SLA1 is reduced during RNAi silencing of the H/ACA RNA binding protein, Cbf5, or pseudouridine synthase.2



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  		FIG. 3.
Primer extension to detect the level of U snRNAs upon silencing Lsm proteins. A, levels of U snRNAs upon Lsm8 silencing. Total RNA (10 µg) from uninduced (–) and induced cells 3 days after adding tetracycline (+) was subjected to primer extension analyses using primers specific to U1, U2, U4, U5, U6, and SLA1 RNA. The level of U3 RNA was used to control the amount of RNA in each sample. B, levels of U snRNAs upon Lsm3 silencing. The analysis was the same as in panel A. snoRNA 92 was used as an internal control for the level of RNA. The extension products were separated on 6% polyacrylamide-7 M urea gel.

 

The Two Lsm Proteins Are Essential for Both trans- and cis-Splicing—Lsm2p-8p complex is essential for splicing in other organisms (9, 38). The effect of Lsm3p and Lsm8p on both trans- and cis-splicing in T. brucei was therefore examined. The results, presented in Fig. 4, A and B, indicate that upon silencing of Lsm8 and Lsm3, the level of SL RNA accumulated and the Y-structure intron was reduced, indicating that trans-splicing was already affected at the first step of splicing. The level of snoRNA-92 was used to control the amount of RNA in different samples.



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  		FIG. 4.
The level of SL RNA and the Y-structure intermediate upon Lsm depletion. A, Lsm8 silencing. RNA (10 µg) from uninduced (–) and induced (+) cells 3 days after tetracycline induction was subjected to primer extension using SL RNA intron-specific primer. The level of snoRNA-92 was used to control for the amount of RNA. B, Lsm3 silencing. Total RNA (10 µg) from unsilenced (–) and silenced (+) cells was subjected to primer extension analysis, as described in panel A. The extension products were separated on 6% polyacrylamide –7 M urea gel and visualized by autoradiography. M, the marker was a pBR322 MspI digest, and the sizes are indicated in nucleotides.

 
Trans-splicing defects are expected to affect the processing of polycistronic pre-mRNA, like that of the tubulin transcript. Indeed, during the silencing of Lsm8 (Fig. 5A) and Lsm3 (Fig. 5B), the level of mature tubulin mRNA was reduced, and the precursor accumulated. To control the level of RNA in each sample, we hybridized the membrane with a 7SL RNA probe. These results further confirm that Lsm3p and Lsm8p are required for trans-splicing in trypanosomes.



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  		FIG. 5.
{alpha}-tubulin mRNA processing during Lsm depletion. A, Lsm8 silencing. RNA (30 µg) from uninduced (–) or induced (+) cells 3 days after tetracycline induction was separated on 1.2% agarose gel. The membrane was hybridized with probes specific to tubulin mRNA and 7SL RNA. A shorter exposure with the tubulin probe is shown on the top, and a longer exposure with the same probe in the middle, demonstrating the accumulation of pre-mRNA. B, Lsm3 silencing. Total RNA (30 µg) was prepared from Lsm3 silenced (+) or unsilenced (–) cells and subjected to Northern analysis. The membrane was hybridized with the same probes as in A.

 
To investigate whether cis-splicing was also affected upon silencing the two proteins, we performed RT-PCR to detect the effects on the level of the PAP transcript, which undergoes both cis- and trans-splicing (26). The results, presented in Fig. 6, indicate that upon silencing of Lsm8 (Fig. 6B, lane 2) and Lsm3 (Fig. 6C, lane 2), the level of mature PAP mRNA is reduced. The pre-PAP mRNA was detected using a sense oligonucleotide specific to the intron region and an antisense oligonucleotide complementary to the second exon (as indicated in Fig. 6A). The results demonstrate that the level of PAP pre-mRNA accumulated in both Lsm8 (Fig. 6B, lane 4) and Lsm3 (Fig. 6C, lane 4) silenced cells. The amount and quality of the template used in RT-PCR was examined by determining the level of snoRNA precursors (Fig. 6B, lanes 5 and 6; Fig. 6C, lanes 7 and 8). The RNA used for RT-PCR was DNA-free, as shown in Fig. 6, B, lanes 7 and 8, and C, lanes 5 and 6. These results suggest that the two proteins are also required for cis-splicing. Taken together, these data indicate the essential role of Lsm3p and Lsm8p in both trans- and cis-splicing in trypanosomes.

Lsm8p Is Required for U4.U6 Dimer and U4.U6/U5 Trimer Formation—It was demonstrated previously (9) that Lsm proteins are required for the formation of functional U6 RNP, U4.U6 dimeric, and U4.U6/U5 trimeric complexes in yeast. To examine whether the silencing of Lsm proteins affects the formation of the different U6-containing complexes in trypanosomes, we examined the distribution of U6-containing RNPs by sucrose gradient fractionation. RNA prepared from sucrose gradient fractions was subjected to Northern analysis. The results, presented in Fig. 7, indicate that upon silencing of Lsm8 (Fig. 7B), the formation of both the U4.U6 dimer and U4.U6/U5 trimer was affected, as compared with unsilenced cells (Fig. 7A). Upon silencing, the level of U6 was dramatically reduced, and the remaining U6 RNA was assembled in the trimeric and larger complex(s) (Fig. 7B, fractions 18–26). No free U6 (fractions 10–12) or U4.U6 dimer (fractions 14–16) was detected. In the silenced cells U4 RNA was shifted toward the top of the gradient, and most of the U4 existed as monomeric U4 RNP (fractions 12–14). In contrast, in uninduced cells most of the U4 RNA was present in the dimeric complex (Fig. 7A, fractions 14–18). In addition, upon silencing the U5 RNA also disappeared from the U4.U6/U5 trimeric complex (fractions 20–22). These results clearly indicate that Lsm8 silencing abolished the formation of U4.U6 dimeric and U4.U6/U5 trimeric complexes.



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  		FIG. 7.
Lsm8p is required for the U4.U6 and U4.U6/U5 complex formation. Whole cell extracts from uninduced (panel A) and induced (panel B) cells were prepared as described under "Experimental Procedures" and fractionated on 10–30% sucrose gradients for 22 h. RNA from even-numbered fractions was subjected to Northern analyses. The membranes were hybridized with probes specific to U4, U5, and U6 snRNAs. The upper panel represents the EtBr staining of the gel, and the hybridization results are shown in the lower panels. The dimer and trimer U6 RNP complexes are indicated. The S-value was determined relative to rRNA 28 S, 18 S, and 4 S (Invitrogen) and Catalase (11 S).

 
Lsm3p and Lsm8p Are Required for mRNA Degradation—In yeast and mammals Lsm8p was shown to exclusively affect U6 stability but not mRNA decay. In contrast, Lsm3p was shown to participate in both U6 and mRNA stability. To examine the effect of silencing these factors on mRNA stability, we treated unsilenced and silenced cells (3 days after induction) with actinomycin D to inhibit transcription and measure the decay rate of mRNAs. RNA was prepared and subjected to Northern analysis to determine the half-life of two mRNAs, the SRP19 (T. brucei genome database accession number Tb10.61.2700) and RHS1 (GenBankTM accession number XP_340234 [GenBank] ) mRNAs. The same membrane was hybridized with an oligonucleotide complementary to 28 S rRNA that serves as an internal control for the level of RNA in each lane. The results are presented in Fig. 8. The level of SRP19 mRNA was measured by densitometry, and the half-life was calculated taking into account the subtle differences in the amount of RNA in the samples as estimated by the level of 28 S rRNA (Fig. 8B). The results indicate that upon silencing of Lsm8 and Lsm3, the SRP19 mRNA was stabilized, because the half-life was increased by 2.1and 1.8-fold, respectively. We further examined the level of RHS1 mRNA, whose half-life was ~30 min.3 Upon silencing of Lsm8 and Lsm3, the half-life of this mRNA was increased by 2.0- and 1.6-fold, respectively, as compared with the uninduced cells (Fig. 8C). These results suggest that like in other organisms, Lsm proteins in trypanosomes are also required for mRNA degradation.



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  		FIG. 8.
Lsm3p and Lsm8p proteins are involved in mRNA decay. Uninduced (Lsm8–Tet) and induced Lsm3 (Lsm3+Tet), as well as induced Lsm8 cells (Lsm8+Tet) 3 days after tetracycline addition were treated with 10 µg/ml actinomycin D at different time points as indicated above the lanes. RNA was prepared from the treated cells and analyzed on 1.2% agarose gel. The membrane was hybridized to detect SRP19, RHS1 mRNAs, and 28 S rRNA. The results are shown in panel A. The hybridized signal was measured by densitometry, and the level of mRNAs was adjusted based on the level of 28 S rRNA present in the samples. The decay curves of SRP19 and RHS1 mRNA are shown in B and C, respectively. The level of mRNA compared with the steady-state level (time point 0) was calculated based on the level of rRNA. The half-life (in min) is indicated.

 

Note that the effect on mRNA decay, as well as on U6 stability, was much more prominent in the Lsm8-silenced cells, as compared with that in the Lsm3-silenced cells. This may stem from more efficient and stable silencing elicited by the stem-loop structure as compared with the T7 opposing system. A great surprise was the finding that the trypanosome Lsm8p functions also in mRNA decay, because in other eukaryotes this protein functions exclusively in U6 RNP biogenesis.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we identified seven Lsm proteins that together constitute the Lsm2p-8p complex. Silencing of Lsm8 and Lsm3 by RNAi indicates that this complex functions in both U6 RNA biogenesis and mRNA decay. The involvement of these proteins in splicing (both trans- and cis-splicing), as well as in mRNA decay, suggests that a single complex may operate in trypanosomes that is amenable to carry out these two different functions.

An inspection of the Sm motifs of the Lsm protein indicated that the Sm motifs 1 and 2 are relatively degenerate and contain only a few highly conserved positions. Several of these positions are also shared with the trypanosome Sm consensus, but Lsm-specific residues also exist (Fig. 1C). As in the case of the trypanosome Sm motifs, there are conservative replacements by similar amino acids. However, some interesting differences do exist. In Sm motif 1 of Lsm2p there is an arginine instead of isoleucine. In Sm 1 of Lsm5p the sequence DDFV present in yeast and mammals is SPTS in trypanosomes, and in Sm motif 2 the sequence GNNI in yeast and mammals is RGSS in trypanosomes, whereas the sequence PGG in yeast and mammals is KHS in trypanosomes. More striking are the differences found in Lsm3p, in which the highly conserved amino acids in Sm 1 differ in the trypanosomes including changes from Asp to (Ala/Val), Leu to Cys, Arg to Thr and Glu to Thr. However, despite these major changes and the deviations from yeast and their human counterparts, this protein still functions in the same processes.

Of special interest is the trypanosome Lsm8p protein, because there is a large insert in the Lsm8p compared with its homologues in other eukaryotes. This insert, however, is within an open reading frame, but the amino acid composition in this open reading frame is highly divergent among the different trypanosomatid species. In searching the EST data base, we found an EST where this sequence was removed, suggesting that this segment may be spliced out. Inspecting the junction of this putative intron did not reveal a consensus sequence of cis-splicing but rather, the same sequence TCGTAAC was found flanking this intron; one of this pair exists in the EST. Most interestingly, this putative intron was not removed when procyclic stage mRNA was used in a RT-PCR assay.4 These data suggest that this intron may be removed only in the bloodstream stage by a non-conventional splicing event, because the EST was derived from bloodstream mRNA. One mechanism that can possibly carry out such splicing removal is an endonuclease/ligase reaction analogous to the reactions that splice the Hac1p pre-mRNA (39).

In mammalian cells the distinction as to which snRNA binds which Sm proteins may be assisted by the polymerases that transcribe these RNAs. The U1, U2, U4, and U5 that are transcribed by RNA polymerase II bind Sm proteins, whereas the U6 that binds to Lsm proteins is transcribed by RNA polymerase III (1). However, in trypanosomes all the U snRNAs are transcribed by polymerase III (27). The SL RNA, however, is transcribed by RNA polymerase II and also binds to Sm proteins (40). It is currently unknown how trypanosome sorts the snRNAs with respect to Sm and Lsm protein binding. This sorting, however, may be assisted by binding of the La protein, which exists in T. brucei (41). Each of the U snRNAs carries a U tail, but most probably La binds only to the U6, and therefore La binding may recruit the Lsm complex. Indeed, it was suggested previously (42, 43) that La "hands over" U6 to the Lsm2p-8p complex, and in the absence of Lsm proteins, La may remain associated with U6 for a longer time.

In this study we provide evidence for the presence of three complexes carrying U6 snRNA in T. brucei (Fig. 7). Previous studies failed to detect the trimeric snRNP complex by native gels (44, 45). However, here we detected distinct complexes carrying the U6 snRNP including the tri-snRNP complex. In the uninduced cells, the U4 peak was ~20 S, which reflects the U4/U6 dimer complex, analogous to the complex described previously (44). Upon silencing of Lsm8, the monomeric U4 RNP appeared in an 18 S peak carrying only U4 but lacking the U6 snRNA. Interestingly, the remaining U6 was found in the tri-snRNP and larger complexes. This finding supports the observation that U5 RNA can form an RNP complex with the U4 and U6 RNAs, as was described previously in T. brucei (46), as well as in L. collosoma (47).

Interestingly, the trypanosome Lsm8p that exclusively functions in U6 RNP biogenesis in other eukaryotes was shown here to associate with U6 RNA and to be required also for mRNA decay. To our surprise, Lsm1p was not identified in the genome data base of T. brucei and T. cruzi. However, we cannot rule out the possibility that such a protein will be discovered when the genome project is completed. Lsm1p is mostly related to Lsm8p in both yeast and humans, and both these proteins are related to SmB (8, 9). The participation of Lsm8p in both U6 stability and mRNA decay may suggest that this protein also bears the characteristics of Lsm1p. If this is indeed the case, there may be only a single Lsm complex in trypanosomes. It is currently unclear how the same complex can function and reside in two distinct compartments, the nucleus, and the cytoplasm. However, if the complex associates with other proteins, such as Xrn1 or Dcp1/2, these proteins can dictate the function and localization of the complex. Indeed, in yeast the association of Lsm8p with an exosomal component, Rrp42p, was demonstrated (48). These data are consistent with the direct association of Lsm2p-Lsm8p with the RNA degradation machinery. On the other hand, the association of Lsm2p-8p with La, a nuclear protein, may direct the complex to interact with U6. The data presented in this study suggest the existence of a single Lsm complex in trypanosomes. Only after purification of the Lsm complex by tagging a protein common to both complexes in other eukaryotes will it be possible to unequivocally determine the number of trypanosome Lsm proteins and to conclude whether a single or two Lsm complexes exist in this organism.

Previous studies suggested that the evolution of Sm proteins proceeded in several steps. The ancestral Sm-like gene, related to the one present in Archaea, would have been duplicated several times to generate the seven different Sm-like proteins. In the second step, these proteins would have been duplicated, leading to the appearance of two distinct, but related subgroups corresponding to the Lsm2p-8p and the canonical Sm complexes found in yeast, humans, and other eukaryotes. Other duplications could have given rise to other Lsm proteins like Lsm1p and Lsm9p. In trypanosomes, which are ancient eukaryotes, maybe the duplication terminated after the generation of a single Sm and Lsm complexes.


   FOOTNOTES
 
* This work was supported by a grant from the Israeli German Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Contributed equally to this study. Back

§ Howard Hughes International Scholar in Molecular Parasitology. To whom correspondence should be addressed. Tel.: 972-3-5318068; Fax: 972-3-5351824; E-mail: michaes{at}mail.biu.ac.il.

1 The abbreviations used are: RNP, ribonucleoprotein; snRNP, small nuclear ribonucleoprotein; SL, spliced leader; dsRNA, double-stranded RNA; RT, reverse transcriptase; EST, expressed sequence tag; snRNA, small nuclear RNA; RNAi, RNA interference; snoRNA, small nucleolar RNA. Back

2 A. Hury, S. Barth, and S. Michaeli, unpublished data. Back

3 E. Ullu, personal communication. Back

4 Q. Liu, X.-h. Liang, S. Uliel, R. Unger, and S. Michaeli, unpublished results. Back



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