Analysis of Spliceosomal Proteins in Trypanosomatids Reveals Novel Functions in mRNA Processing

Itai Dov Tkacz,Sachin Kumar Gupta,Vadim Volkov,Mali Romano,Tomer Haham,Pawel Tulinski,Ilana Lebenthal,Shulamit Michaeli

doi:10.1074/jbc.m109.095349

Abstract

In trypanosomatids, all mRNAs are processed via trans-splicing, although cis-splicing also occurs. In trans-splicing, a common small exon, the spliced leader (SL), which is derived from a small SL RNA species, is added to all mRNAs. Sm and Lsm proteins are core proteins that bind to U snRNAs and are essential for both these splicing processes. In this study, SmD3- and Lsm3-associated complexes were purified to homogeneity from Leishmania tarentolae. The purified complexes were analyzed by mass spectrometry, and 54 and 39 proteins were purified from SmD3 and Lsm complexes, respectively. Interestingly, among the proteins purified from Lsm3, no mRNA degradation factors were detected, as in Lsm complexes from other eukaryotes. The U1A complex was purified and mass spectrometry analysis identified, in addition to U1 small nuclear ribonucleoprotein (snRNP) proteins, additional co-purified proteins, including the polyadenylation factor CPSF73. Defects observed in cells silenced for U1 snRNP proteins suggest that the U1 snRNP functions exclusively in cis-splicing, although U1A also participates in polyadenylation and affects trans-splicing. The study characterized several trypanosome-specific nuclear factors involved in snRNP biogenesis, whose function was elucidated in Trypanosoma brucei. Conserved factors, such as PRP19, which functions at the heart of every cis-spliceosome, also affect SL RNA modification; GEMIN2, a protein associated with SMN (survival of motor neurons) and implicated in selective association of U snRNA with core Sm proteins in trypanosomes, is a master regulator of snRNP assembly. This study demonstrates the existence of trypanosomatid-specific splicing factors but also that conserved snRNP proteins possess trypanosome-specific functions.

Highlights

International Research Scholar grant from the Howard Hughes Medical Institute
The identity of the RNAs enriched in the purification of both SmD3 and Lsm3 was verified by primer extension using U snRNA-specific probes for SmD3 purification (Fig. 1D, panel a) and for Lsm3 purification (Fig. 1D, panel b)
The Sm, Lsm, and U1A complexes were purified from Leishmania

Summary

Lsm and Sm Proteins in Trypanosomatids

Two recent studies purified and analyzed Trypanosoma brucei snRNP proteins by mass spectrometry. Functional studies on two of these proteins, Lsm and Lsm, suggest that these proteins bind U6 and affect mRNA stability [11]. The SmD3-, Lsm3-, and U1A-associated proteins were purified from Leishmania tarentolae and subjected to mass spectrometry. The function of selected snRNP proteins that were identified by mass spectrometry in Leishmania was elucidated by RNAi silencing and in situ tagging in T. brucei. The purification of U1A-associated proteins from L. tarentolae identified, in addition to the U1 snRNP proteins, factors involved in splicing and polyadenylation. PRP19, a splicing factor that is associated with U5 snRNP in active cis-spliceosomes [27], affected the capping of SL RNA and is found in the SL RNP factory, suggesting a unique function in trypanosomes. This study highlights the function of universal splicing factors that acquired unique functions in trypanosomes, as well as that of trypanosome-specific splicing factors

EXPERIMENTAL PROCEDURES

RESULTS

Hypothetical proteins

RNA processing factors

Translation factors

DISCUSSION