Cyclin-Dependent Kinase CRK9, Required for Spliced Leader trans Splicing of Pre-mRNA in Trypanosomes,Functions in a Complex with a New L-Type Cyclin and a Kinetoplastid-Specific Protein

In eukaryotes, cyclin-dependent kinases (CDKs) control the cell cycle and critical steps in gene expression. The lethal parasite Trypanosoma brucei, member of the phylogenetic order Kinetoplastida, possesses eleven CDKs which, due to high sequence divergence, were generically termed CDC2-related kinases (CRKs). While several CRKs have been implied in the cell cycle, CRK9 was the first trypanosome CDK shown to control the unusual mode of gene expression found in kinetoplastids. In these organisms, protein-coding genes are arranged in tandem arrays which are transcribed polycistronically. Individual mRNAs are processed from precursor RNA by spliced leader (SL) trans splicing and polyadenylation. CRK9 ablation was lethal in cultured trypanosomes, causing a block of trans splicing before the first transesterification step. Additionally, CRK9 silencing led to dephosphorylation of RNA polymerase II and to hypomethylation of the SL cap structure. Here, we tandem affinity-purified CRK9 and, among potential CRK9 substrates and modifying enzymes, discovered an unusual tripartite complex comprising CRK9, a new L-type cyclin (CYC12) and a protein, termed CRK9-associated protein (CRK9AP), that is only conserved among kinetoplastids. Silencing of either CYC12 or CRK9AP reproduced the effects of depleting CRK9, identifying these proteins as functional partners of CRK9 in vivo. While mammalian cyclin L binds to CDK11, the CRK9 complex deviates substantially from that of CDK11, requiring CRK9AP for efficient CRK9 complex formation and autophosphorylation in vitro. Interference with this unusual CDK rescued mice from lethal trypanosome infections, validating CRK9 as a potential chemotherapeutic target.     

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

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.     

The spliceosomal PRP19 complex of trypanosomes

In trypanosomes, mRNAs are processed by spliced leader (SL) trans splicing, in which a capped SL, derived from SL RNA, is spliced onto the 5′ end of each mRNA. This process is mediated by the spliceosome, a large and dynamic RNA‐protein machinery consisting of small nuclear ribonucleoproteins (snRNPs) and non‐snRNP proteins. Due to early evolutionary divergence, the amino acid sequences of trypanosome splicing factors exhibit limited similarity to those of their eukaryotic orthologs making their bioinformatic identification challenging. Most of the ～ 60 protein components that have been characterized thus far are snRNP proteins because, in contrast to individual snRNPs, purification of intact spliceosomes has not been achieved yet. Here, we characterize the non‐snRNP PRP19 complex of Trypanosoma brucei. We identified a complex that contained the core subunits PRP19, CDC5, PRL1, and SPF27, as well as PRP17, SKIP and PPIL1. Three of these proteins were newly annotated. The PRP19 complex was associated primarily with the activated spliceosome and, accordingly, SPF27 silencing blocked the first splicing step. Interestingly, SPF27 silencing caused an accumulation of SL RNA with a hypomethylated cap that closely resembled the defect observed previously upon depletion of the cyclin‐dependent kinase CRK9, indicating that both proteins may function in spliceosome activation.     

Structure-function analysis of the trypanosomatid spliced leader RNA.

In trypanosomes, all mRNAs possess a spliced leader (SL) at their 5' end. SL is added to pre-mRNA via trans -splicing from a small RNA, the SL RNA. To examine structure-function aspects of the trypanosomatid SL RNA, an in vivo system was developed in the monogenetic trypanosomatid Leptomonas collosoma to analyze the function of chimeric and site-directed SL RNA mutants in trans -splicing. Stable cell lines expressing chimeric and mutated SL RNA from the authentic SL RNA regulatory unit were obtained. The chimeric RNA was expressed and assembled into an SL RNP particle, but could not serve as a substrate in splicing. Mutations in loop II and III of L.collosoma SL RNA formed the Y structure intermediate. In addition, a double SL RNA mutant in loop II, and positions 7 and 8 of the intron, also formed the Y structure intermediate, suggesting that these intron positions, although proposed to participate in the interaction of SL RNA with U5, may not be crucial for the first step of the trans -splicing reaction. A mutation in the exon located in loop I was not utilized in splicing, suggesting the importance of exon sequences for trans -splicing in trypanosomes. However, a double SL RNA mutant in loop II and exon position 31 was utilized in both steps of splicing; the mutant thus provides a model molecule for further analysis of positions essential for the function of the SL RNA.     

Characterization of RNase R-digested cellular RNA source that consists of lariat and circular RNAs from pre-mRNA splicing

Besides linear RNAs, pre-mRNA splicing generates three forms of RNAs: lariat introns, Y-structure introns from trans-splicing, and circular exons through exon skipping. To study the persistence of excised introns in total cellular RNA, we used three Escherichia coli 3′ to 5′ exoribonucleases. Ribonuclease R (RNase R) thoroughly degrades the abundant linear RNAs and the Y-structure RNA, while preserving the loop portion of a lariat RNA. Ribonuclease II (RNase II) and polynucleotide phosphorylase (PNPase) also preserve the lariat loop, but are less efficient in degrading linear RNAs. RNase R digestion of the total RNA from human skeletal muscle generates an RNA pool consisting of lariat and circular RNAs. RT–PCR across the branch sites confirmed lariat RNAs and circular RNAs in the pool generated by constitutive and alternative splicing of the dystrophin pre-mRNA. Our results indicate that RNase R treatment can be used to construct an intronic cDNA library, in which majority of the intron lariats are represented. The highly specific activity of RNase R implies its ability to screen for rare intragenic trans-splicing in any target gene with a large background of cis-splicing. Further analysis of the intronic RNA pool from a specific tissue or cell will provide insights into the global profile of alternative splicing.     

Antigenic variation in trypansosomes: Secrets surface slowly

Among pathogenic micro-organisms that evade the mammalian immune responses, Trypanosoma brucei has developed the most elaborate capacity for antigenic variation. Trypanosomes branched early during eukaryotic evolution. They are characterized by many aberrations, ranging from the unusual compartmentation of metabolic pathways to the heresy of RNA editing. The ubiquitous phenomenon of glycosylphosphatidylinositol-anchoring of eukaryotic plasma membrane proteins and RNA trans-splicing (trypanosome genes contain no introns), which adds an identical leader sequence to all trypanosome mRNAs, were first defined during studies of antigenic variation. Genetic transformation of trypanosomes and the high efficiency of gene targeting provide new opportunities to investigate the regulation of antigenic variation. There is every reason to expect trypanosomes to provide further surprises and insights into the evolution of genetic regulatory mechanisms.     

The Octatricopeptide Repeat Protein Raa8 Is Required for Chloroplast trans Splicing

The mRNA maturation of the tripartite chloroplast psaA gene from the green alga Chlamydomonas reinhardtii depends on various nucleus-encoded factors that participate in trans splicing of two group II introns. Recently, a multiprotein complex was identified that is involved in processing the psaA precursor mRNA. Using coupled tandem affinity purification (TAP) and mass spectrometry analyses with the trans-splicing factor Raa4 as a bait protein, we recently identified a multisubunit ribonucleoprotein (RNP) complex comprising the previously characterized trans-splicing factors Raa1, Raa3, Raa4, and Rat2 plus novel components. Raa1 and Rat2 share a structural motif, an octatricopeptide repeat (OPR), that presumably functions as an RNA interaction module. Two of the novel RNP complex components also exhibit a predicted OPR motif and were therefore considered potential trans-splicing factors. In this study, we selected bacterial artificial chromosome (BAC) clones encoding these OPR proteins and conducted functional complementation assays using previously generated trans-splicing mutants. Our assay revealed that the trans-splicing defect of mutant F19 was restored by a new factor we named RAA8; molecular characterization of complemented strains verified that Raa8 participates in splicing of the first psaA group II intron. Three of six OPR motifs are located in the C-terminal end of Raa8, which was shown to be essential for restoring psaA mRNA trans splicing. Our results support the important role played by OPR proteins in chloroplast RNA metabolism and also demonstrate that combining TAP and mass spectrometry with functional complementation studies represents a vigorous tool for identifying trans-splicing factors.     

An in vivo selection method to optimize trans-splicing ribozymes

Group I intron ribozymes can repair mutated mRNAs by replacing the 3′-terminal portion of the mRNA with their own 3′-exon. This trans-splicing reaction has the potential to treat genetic disorders and to selectively kill cancer cells or virus-infected cells. However, these ribozymes have not yet been used in therapy, partially due to a low in vivo trans-splicing efficiency. Previous strategies to improve the trans-splicing efficiencies focused on designing and testing individual ribozyme constructs. Here we describe a method that selects the most efficient ribozymes from millions of ribozyme variants. This method uses an in vivo rescue assay where the mRNA of an inactivated antibiotic resistance gene is repaired by trans-splicing group I intron ribozymes. Bacterial cells that express efficient trans-splicing ribozymes are able to grow on medium containing the antibiotic chloramphenicol. We randomized a 5′-terminal sequence of the Tetrahymena thermophila group I intron and screened a library with 9 × 10⁶ ribozyme variants for the best trans-splicing activity. The resulting ribozymes showed increased trans-splicing efficiency and help the design of efficient trans-splicing ribozymes for different sequence contexts. This in vivo selection method can now be used to optimize any sequence in trans-splicing ribozymes.     

Regulation of mammalian pre-mRNA splicing

In eukaryotes, most protein-coding genes contain introns which are removed by precursor messenger RNA (pre-mRNA) splicing. Alternative splicing is a process by which multiple messenger RNAs (mRNAs) are generated from a single pre-mRNA, resulting in functionally distinct proteins. Recent genome-wide analyses of alternative splicing indicated that in higher eukaryotes alternative splicing is an important mechanism that generates proteomic complexity and regulates gene expression. Mis-regulation of splicing causes a wide range of human diseases. This review describes the current understanding of pre-mRNA splicing and the mechanisms that regulate mammalian pre-mRNA splicing. It also discusses emerging directions in the field of alternative splicing. Supported by the Program of “one Hundred Talented people” of the Chinese Academy of Sciences.