Novel structure of a human U6 snRNA pseudogene

A genomic DNA library containing human placental DNA cloned into phage lambda Charon 4A was screened for snRNA U6 genes. In vitro 32P-labeled U6 snRNA isolated from HeLa cells was used as a hybridization probe. A positive clone containing a 4.6-kb EcoRI fragment of human chromosomal DNA was recloned into the EcoRI site of pBR325 and mapped by restriction endonuclease digestion. Restriction fragments containing U6 RNA sequences were identified by hybridization with isolated U6[32P]RNA. The sequence analysis revealed a novel structure of a U6 RNA pseudogene, bearing two 17-nucleotide(nt)-long direct repeats of genuine U6 RNA sequences arranged in a head-to-tail fashion within the 5' part of the molecule. Hypothetical models as to how this type of snRNA U6 pseudogene might have been generated during evolution of the human genome are presented. When compared to mammalian U6 RNA sequences the pseudogene accounts for a 77% overall sequence homology and contains the authentic 5'- and 3'-ends of the U6 RNA.     

Structure and expression of the U5 snRNA gene of Arabidopsis thaliana. Conserved upstream sequence elements in plant U-RNA genes.

We have previously characterized the U2 small nuclear (sn) RNA gene family of Arabidopsis thaliana. To find out the structural features of upstream and downstream non-coding regions that are shared by different U-RNA genes in higher plants we have isolated the gene encoding a 125 nt-long U5 snRNA of Arabidopsis. Activity of the cloned gene was demonstrated in stably transformed tobacco calli and by transient expression in transfected protoplasts of Nicotiana plumbaginifolia. Southern analysis indicated that the Arabidopsis genome contains 8-9 copies of the U5 gene. Alignment of upstream non-coding regions revealed two elements conserved between all plant U-RNA genes characterized so far: the sequence RTCCCACATCG (-70/-80 region, 100% conservation) and the TATA homology around position -30. The coding regions in all genes are followed by the sequence CAN4-9AGTN (A/T)AA which may correspond to a termination and/or processing signal.     

Analysis of a gene cluster coding for the Xenopus laevis U7 snRNA.

A cluster of Xenopus laevis U7 snRNA genes has been isolated and sequenced. The gene structure is more compact than, but otherwise comparable to, the major U snRNA genes since the distal sequence element (DSE) is located only 4 nt upstream of the PSE. The corresponding RNA is present in the oocyte and accumulates early in oogenesis.     

Conserved sequences in the U2 snRNA-encoding genes of Kinetoplastida do not include the putative branchpoint recognition region

The U2 small nuclear RNA (snRNA) of Trypanosoma brucei gambiense, a flagellated protozoon of the order Kinetoplastida, is 148 nucleotides (nt) long, and thus the smallest U2 snRNA identified so far. To examine the evolutionary conservation of this RNA among Kinetoplastida, we have cloned and sequenced the U2 genes from Trypanosoma congolense and Leishmania mexicana amazonensis, which are 145 and 141 nt in length, respectively. The sequences of the Kinetoplastida U2 snRNAs are essentially identical in the 5' half of the molecule. Surprisingly, the putative branch site recognition sequence of L. m. amazonensis U2 snRNA shows two nt changes when compared with the other two U2 snRNAs. The sequence of the 3' half of the Kinetoplastida U2 snRNAs is less conserved with T. congolense and L. m. amazonensis RNAs showing 23 and 35 nt sequence variations, respectively, when compared with the corresponding sequence of the T. b. gambiense U2 snRNA. Alignment of the flanking regions of the U2 genes revealed several elements which are conserved both in sequence and in position relative to the U2 coding region and which may function in the biosynthesis of U2 snRNAs. One upstream element specifically binds protein factor(s) present in T. brucei nuclear extracts.     

Activity of chimeric RNAs of U6 snRNA and (-)sTRSV in the cleavage of a substrate RNA.

U6 small nuclear RNA is one of the spliceosomal RNAs essential for pre-mRNA splicing. Discovery of mRNA-type introns in the highly conserved region of the U6 snRNA genes led to the hypothesis that U6 snRNA functions as a catalytic element during pre-mRNA splicing. The highly conserved region of U6 snRNA has a structural similarity with the catalytic domain of the negative strand of the satellite RNA of tobacco ring spot virus [(-)sTRSV], suggesting that the highly conserved region of U6 snRNA forms the catalytic center. We examined whether synthetic RNAs consisting of the sequence of the highly conserved region of U6 snRNA or various chimeric RNAs between the U6 region and the catalytic RNA of (-)sTRSV could cleave a substrate RNA that can partially base-pair with them and have a GU sequence. Chimeric RNAs with 70 to 83% sequence identity with the conserved region of S. pombe U6 snRNA cleaved the substrate RNA at the 5' side of the GU sequence, which is shared by the 5' end of an intron in a pre-mRNA. We found that the highly conserved region of U6 snRNA and the catalytic domain of (-)sTRSV are strikingly similar in structure to the catalytic core region of the group I self-splicing intron in cyanobacteria. These results suggest that U6 snRNA, (-)sTRSV and the group I self-splicing intron originated from a common ancestral RNA, and support the hypothesis that U6 snRNA catalyzes pre-mRNA splicing reaction.     

水稻U2snRNA基因的分离及结构分析

对水稻(Oryza sativa L.)基因文库中分离到的U2snRNA基因FDRGU2.3进行序列分析,其编码区与小麦(Triticum aestivum L、)、玉米(Zea mays L.)、豌豆(Pisum sativum L.)及拟南芥(Arabidopsis thaliana(L.)Heyhy.)等植物U2基因的同源性均大于80％,且5＇端70个碱基高度保守。在基因编码区上游-70及-30区分别包含有植物UsnRNA基因特有的上游顺序元件(USE)及类TATA元件。同其它植物一样,水稻U2.3snRNA的二级结构也有保守的4个茎环区。其中环Ⅱ的结构与单子叶植物中的小麦和玉米相同,但与双子叶植物的豌豆和拟南芥存在明显差异。环Ⅳ的结构在单子叶和双子叶植物中亦有不同的变化。这些差异可能意味着单子叶和双子叶植物的剪接机构有所区别。