The Trypanosoma brucei La protein is a candidate poly(U) shield that impacts spliced leader RNA maturation and tRNA intron removal

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Abstract

By virtue of its preferential binding to poly(U) tails on small RNA precursors and nuclear localisation motif, the La protein has been implicated for a role in the stabilisation and nuclear retention of processing intermediates for a variety of small RNAs in eukaryotic cells. As the universal substrate for trans-splicing, the spliced leader RNA is transcribed as a precursor with just such a tail. La protein was targeted for selective knockdown by inducible RNA interference in Trypanosoma brucei. Of three RNA interference strategies employed, a p2T7-177 vector was the most effective in reducing both the La mRNA as well as the protein itself from induced cells. In the relative absence of La protein T. brucei cells were not viable, in contrast to La gene knockouts in yeast. A variety of potential small RNA substrates were examined under induction, including spliced leader RNA, spliced leader associated RNA, the U1, U2, U4, and U6 small nuclear RNAs, 5S ribosomal RNA, U3 small nucleolar RNA, and tRNATyr. None of these molecules showed significant variance in size or abundance in their mature forms, although a discrete subset of intermediates appear for spliced leader RNA and tRNATyr intron splicing under La depletion conditions. 5′-end methylation in the spliced leader RNA and U1 small nuclear RNA was unaffected. The immediate cause of lethality in T. brucei was not apparent, but may represent a cumulative effect of multiple defects including processing of spliced leader RNA, tRNATyr and other unidentified RNA substrates. This study indicates that La protein binding is not essential for maturation of the spliced leader RNA, but does not rule out the presence of an alternative processing pathway that could compensate for the absence of normally-associated La protein.

Introduction

The 5′ end of all nuclear-derived mRNAs in kinetoplastid protozoa consists of a 39-nt spliced leader (SL) derived from a precursor transcript, the SL RNA (Liang et al., 2003). Trans-splicing of the SL represents the first step in the maturation of a polycistronic pre-mRNA to the mature capped and polyadenylated mRNA (LeBowitz et al., 1993, Matthews et al., 1994). The mechanism of trans-splicing closely resembles the mechanism of cis-splicing and uses similar small nuclear (sn) RNA and protein components (Tschudi and Ullu, 1990, Lücke et al., 1997). The SL RNA is a bifunctional transcript unique to trans-splicing. It is characterised by a phylogenetically-conserved secondary structure (Bruzik et al., 1988) and is found in a ribonucleoprotein (RNP) complex (Miller and Wirth, 1988). The 3′ end, or intron, of the SL RNA resembles a splicing-associated snRNA, while the 5′ end, the SL, is the common 5′ substrate in the splicing reaction that provides the mature mRNAs with a hypermethylated cap (Bangs et al., 1992) and a pseudouridine modification (Liang et al., 2002). Assembly of a functional SL RNP requires the addition of core and specific protein components to an SL RNA that has been trimmed and modified from the primary transcript (Ismaïli et al., 1999, Palfi et al., 2000). The SL RNA precursor possesses a 3′ poly(U) tail (Sturm et al., 1999) that is removed in an Sm protein-dependent manner (Mandelboim et al., 2003, Zeiner et al., 2004a) after transport to the cytoplasm (Zeiner et al., 2003a).

Removal of the poly(U) tail for the SL RNA precursor appears to be a two-step process (Zeiner et al., 2004b). We propose that intermediate polyuridylated forms of the SL RNA are protected from 3′-exonucleolytic degradation by a ‘poly(U)-shield’ protein, consistent with the observations that the poly(U) tail is neither removed prior to nuclear egress (Zeiner et al., 2003a), nor before Sm-protein binding (Sturm et al., 1999). The most-abundant protein that performs such poly(U)-shielding function in eukaryotic cells is the homolog of the La autoantigen (Stefano, 1984, Maraia and Intine, 2002, Wolin and Cedervall, 2002). The La protein, which is dispensible for growth in two yeast genera (Yoo and Wolin, 1994, Van Horn et al., 1997), performs diverse functions including: transcription initiation and termination by RNA polymerase (pol) III (Maraia et al., 1994); binding to most nascent RNA pol III transcripts such as 5S rRNA and tRNA, where it protects precursor molecules (Preiser et al., 1993) and is necessary for tRNA maturation, like pre-tRNA folding (Chakshusmathi et al., 2003) and 3′ processing (Kufel and Tollervey, 2003); assembly of pol II- and pol III-transcribed snRNAs into snRNPs where it acts as a chaperone (Pannone et al., 1998, Xue et al., 2000); association with vault and telomerase complexes (Aigner et al., 2000, Kickhoefer et al., 2002); cap-dependent (Cardinali et al., 2003) and cap-independent Internal Ribosome Entry Site (IRES)-mediated (Pudi et al., 2003) translation of mRNA; and transposon mobility (Aye and Sandmeyer, 2003). If La protein acts as a poly(U) shield for precursor SL RNA, its absence could manifest itself in several molecular phenotypes: (i) SL RNA 3′-end maturation may be overzealous and/or occur in an incorrect temporal order, resulting in 3′-overexpressing with a possible loss of 5′ cap 4 formation; (ii) maturation may be slowed, resulting in the accumulation of 3′-extended SL RNA; or (iii) an alternative pathway may compensate for the absence of La, revealing no visible effect on SL RNA.

The gene for the La protein ortholog has been identified in Trypanosoma brucei (Marchetti et al., 2000, Westermann and Weber, 2000). Recombinant T. brucei La protein has been shown to bind poly(U) in vitro (Westermann and Weber, 2000, Dong et al., 2004) and the crystal structure of the La motif has been determined (Dong et al., 2004). To test whether the kinetoplastid La protein is involved in the SL RNA maturation pathway we have tested three different double-stranded RNA-interference (RNAi) plasmids for effects of La protein knockdown on a variety of polyuridylated small RNAs. In contrast to the yeasts, knockdown of La protein with the p2T7-177 vector yielded a lethal phenotype in T. brucei.

Section snippets

Plasmid constructions

Three vectors for RNAi were constructed as follows. The 3′ part of the La gene spanning nts 315-1004 was amplified by PCR from T. brucei 427 genomic DNA using oligonucleotides LaF1 (5′-CCCACAGCAACACTCGAGCA) and LaR1 (5′-CCCTCTAGATTCACGTGACCGCTTGTGTC) or LaR2 (5′-CCCGGATCCTTCACGTGACCGCTTGTGTC), with encoded Xho I and added Xba I or Bam HI recognition sites underlined. The amplified fragments were called La-A (product of LaF1 and LaR1 PCR) and La-B (product of LaF1 and LaR2 PCR) and were cloned

RNAi effects vary by vector

Three plasmids for inducible RNAi were used to knockdown the expression of La protein due to empirical differences found for other targets. pLew100, pZJM and p2T7-177 were chosen as representative vectors with variation in transcription strategies and/or integration site targeting: pLew100 uses the T. brucei PARP promoter to transcribe a single transcript containing two opposing fragments of the target gene separated by a stuffer sequence and integrates into the rRNA locus, while pZJM and

Discussion

We examined the role of La protein in the maturation of small RNA molecules. Using RNAi to reduce the presence of La mRNA and protein in T. brucei procyclic cells, we show an anticipated effect on the removal of an intron located in tRNATyr analogous to the splicing inhibition seen for tRNAs in other systems upon La knockout. Superficially, no phenotypic changes were evident in a host of other potential RNA substrates containing poly(U) stretches at their 3′ ends, however, the SL RNA showed

Acknowledgements

We thank Paul T. Englund, Keith Gull, Klaus Ersfeld, and Kent L. Hill for providing RNAi vectors, Paul A.M. Michels for providing the GAPDH antisera, and Gus M. Zeiner and members of our laboratories for stimulating discussions and helpful comments. This work was supported by NIH grants AI34536 and AI056034 to NRS and DAC, and by grants A5022302 and Z60220518 from the Grant Agency of the Czech Academy of Sciences to JL.

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