Sequences required for 3' end formation of human U2 small nuclear RNA

Xenopus oocytes injected with human U2 snRNA genes synthesize mature U2 as well as a U2 precursor with about 10 extra 3' nucleotides (human pre-U2 RNA). Formation of the pre-U2 3' end requires a downstream element located between position +16 and +37 in the U2 3'-flanking sequence. The distance between this element and the U2 coding region can be increased without affecting formation of the pre-U2 3' end. When the natural sequence surrounding the pre-U2 3' end is changed, novel 3' ends are still generated within a narrow range upstream from the element. The 3' terminal stem-loop of U2 snRNA is not required for pre-U2 3' end formation. A sequence within the 3' element (GTTTN0-3AAAPuNNAGA) is conserved among snRNA genes transcribed by RNA polymerase II. Our results suggest that the 3' ends of pre-U2 RNA and histone mRNA may be generated by related but distinct RNA processing mechanisms.     

专一切割苹果锈果类病毒多体自切割核酶的克隆和转录物的体外活性测定

将苹果锈果类病毒的1个14nt的靶序列连接在锤头型核酶的3′末端,构成自切割核酶。经人工合成和PCR扩增,克隆在转录载体pGEM7zf（＋）的XhoⅠ-Hind Ⅲ位点。利用限制酶Xho I与SalI的连接,消失其识别位点序列,将自切割核酶片段插入到重组质粒中,经连续5次亚克隆,分别获得2、4、6、8、10和12拷贝的多体自切割核酶。在T7RNA聚合酶作用下,线性化重组质粒转录的多体自切割核酶通过内部的顺式切割释放出较多数量的核酶分子,提示在转录水平能够提高核酶转录物的浓度。用相同摩尔浓度的单体和12体自切割核酶分别对32P标记的靶ASSVd进行反式切割,核酶与靶RNA摩尔浓度比为1：1。放射自显影结果表明：多体自切割核酶对靶ASSVd的切割效率明显高于单体自切割核酶。我们推测多体自切割核酶在体内系统中可能具有更好的应用价值。     

Mutational analysis of an essential binding site for the U3 snoRNA in the 5' external transcribed spacer of yeast pre-rRNA.

The small nucleolar RNA U3 is essential for viability in yeast. We have previously shown that U3 can be cross-linked in vivo to the pre-rRNA in the 5' external transcribed spacer (ETS), at +470. This ETS region contains 10 nucleotides of perfect complementarity to U3. In a genetic background where the mutated rDNA is the only transcribed rDNA repeat, the deletion of the 10 nt complementary to U3 is lethal. Cells lacking the U3 complementary sequence in pre-rRNA fail to accumulate 18S rRNA: pre-rRNA processing is inhibited at sites A0 in the 5' ETS, A1 at the 5' end of 18S rRNA and A2 in ITS1. We show here that effects on processing at site A0 are specific for U3 and its associated proteins and are not seen on depletion of other snoRNP components. The deletion of the sequence complementary to U3 in the ETS therefore mimics all the known effects of the depletion of U3 in trans. This indicates that we have identified an essential U3 binding site on pre-rRNA, required in cis for the maturation of 18S rRNA.     

核酶对bcl－2mRNA的体外切割

通过微机对ｂｃｌ－２ＲＮＡ二级结构的分析,设计针对ｂｃｌ－２片段５＇ＣＧＣＧＡＣＣＣＧＧＵＣＧＣＣＡＧＧＡＣＣＵＣＧ３＇的“锤头状”（Ｈａｍｍｅｒｈｅａｄ）核酶（Ｒｉｂｏｚｙｍｅ,ＲＤ）基因,平端连接于ｐＧＥＭ－３Ｚｆ（－）ＨｉｎｃⅡ位点,克隆后经测序表明序列正确,ｂｃｌ－２和Ｒｉｂｏｚｙｍｅ基因经体外转录,５０℃作用２ｈ,从１６５６－１６５７（Ｃ－Ｇ）位之间切断ｂｃｌ－２ＲＮＡ片段．     

Formation of the 3'' end of U1 snRNA is directed by a conserved sequence located downstream of the coding region.

U1 is a small non-polyadenylated nuclear RNA that is transcribed by RNA polymerase II and is known to play a role in mRNA splicing. The mature 3' end of U1 snRNA is formed in at least two steps. The first step generates precursors of U1 RNA with a few extra nucleotides at the 3' end; in the second step, these precursors are shortened to mature U1 RNA. Here, I have determined the sequences required for the first step. Human U1 genes with various deletions and substitutions near the 3' end of the coding region were constructed and introduced into HeLa cells by DNA transfection. The structure of the RNA synthesized during transient expression of the exogenous U1 gene was analyzed by S1 mapping. The results show that a 13 nucleotide sequence located downstream from the U1 coding region and conserved among U1, U2 and U3 genes of different species is the only sequence required to direct the first step in the formation of the 3' end of U1 snRNA.     

Mutational analysis of the promoter required for influenza virus virion RNA synthesis.

An in vitro RNA synthesis system was established in which the influenza virus virion (minus-sense) RNA was made from the synthetic plus-sense RNA (cRNA) template by the purified viral polymerase complex. The cRNA promoter was studied by mutational analysis using the in vitro system, and on the basis of these experiments, the first 11 nucleotides of the 3' noncoding sequence were found to contain the minimum promoter required for virion RNA synthesis. The addition of extra nucleotides at the 3' end decreased the promoter activity of the templates, indicating that the viral polymerase does not recognize an internal promoter efficiently. The wild-type and mutated RNA templates were also tested in vivo by using the ribonucleoprotein transfection system. In contrast to the in vitro system, it was found that the majority of mutations at the 3'-terminal sequence significantly decreased or abolished chloramphenicol acetyltransferase (CAT) expression. These results suggest that the cRNA promoter overlaps other essential cis elements required for chloramphenicol acetyltransferase expression in vivo.