Zamudio Jesse R, Mittra Bidyottam, Chattopadhyay Arnab, Wohlschlegel James A, Sturm Nancy R, Campbell David A
Department of Microbiology, Immunology, & Molecular Genetics, University of California at Los Angeles, Los Angeles, CA 90095-1489, USA.
Mol Cell Biol. 2009 Mar;29(5):1202-11. doi: 10.1128/MCB.01496-08. Epub 2008 Dec 22.
Kinetoplastid flagellates attach a 39-nucleotide spliced leader (SL) upstream of protein-coding regions in polycistronic RNA precursors through trans splicing. SL modifications include cap 2'-O-ribose methylation of the first four nucleotides and pseudouridine (psi) formation at uracil 28. In Trypanosoma brucei, TbMTr1 performs 2'-O-ribose methylation of the first transcribed nucleotide, or cap 1. We report the characterization of an SL RNA processing complex with TbMTr1 and the SLA1 H/ACA small nucleolar ribonucleoprotein (snoRNP) particle that guides SL psi(28) formation. TbMTr1 is in a high-molecular-weight complex containing the four conserved core proteins of H/ACA snoRNPs, a kinetoplastid-specific protein designated methyltransferase-associated protein (TbMTAP), and the SLA1 snoRNA. TbMTAP-null lines are viable but have decreased SL RNA processing efficiency in cap methylation, 3'-end maturation, and psi(28) formation. TbMTAP is required for association between TbMTr1 and the SLA1 snoRNP but does not affect U1 small nuclear RNA methylation. A complex methylation profile in the mRNA population of TbMTAP-null lines indicates an additional effect on cap 4 methylations. The TbMTr1 complex specializes the SLA1 H/ACA snoRNP for efficient processing of multiple modifications on the SL RNA substrate.
动质体鞭毛虫通过反式剪接将一个39个核苷酸的剪接前导序列（SL）连接到多顺反子RNA前体中蛋白质编码区的上游。SL修饰包括前四个核苷酸的帽2'-O-核糖甲基化以及尿嘧啶28处假尿嘧啶（ψ）的形成。在布氏锥虫中，TbMTr1对第一个转录的核苷酸，即帽1进行2'-O-核糖甲基化。我们报道了一种含有TbMTr1和指导SL ψ(28)形成的SLA1 H/ACA小核仁核糖核蛋白（snoRNP）颗粒的SL RNA加工复合体的特征。TbMTr1存在于一个高分子量复合体中，该复合体包含H/ACA snoRNPs的四个保守核心蛋白、一种名为甲基转移酶相关蛋白（TbMTAP）的动质体特异性蛋白以及SLA1 snoRNA。TbMTAP缺失的细胞系是有活力的，但在帽甲基化、3'端成熟和ψ(28)形成方面，SL RNA加工效率降低。TbMTAP是TbMTr1与SLA1 snoRNP之间结合所必需的，但不影响U1小核RNA甲基化。TbMTAP缺失细胞系的mRNA群体中的复杂甲基化图谱表明对帽4甲基化还有额外影响。TbMTr1复合体使SLA1 H/ACA snoRNP专门用于对SL RNA底物进行多种修饰的高效加工。
