Price J V, Engberg J, Cech T R
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder 80309.
J Mol Biol. 1987 Jul 5;196(1):49-60. doi: 10.1016/0022-2836(87)90510-9.
The intervening sequence (IVS) of the Tetrahymena thermophila ribosomal RNA precursor undergoes accurate self-splicing in vitro. The work presented here examines the requirement for Tetrahymena rRNA sequences in the 5' exon for the accuracy and efficiency of splicing. Three plasmids were constructed with nine, four and two nucleotides of the natural 5' exon sequence, followed by the IVS and 26 nucleotides of the Tetrahymena 3' exon. RNA was transcribed from these plasmids in vitro and tested for self-splicing activity. The efficiency of splicing, as measured by the production of ligated exons, is reduced as the natural 5' exon sequence is replaced with plasmid sequences. Accurate splicing persists even when only four nucleotides of the natural 5' exon sequence remain. When only two nucleotides of the natural exon remain, no ligated exons are observed. As the efficiency of the normal reaction diminishes, novel RNA species are produced in increasing amounts. The novel RNA species were examined and found to be products of aberrant reactions of the precursor RNA. Two of these aberrant reactions involve auto-addition of GTP to sites six nucleotides and 52 nucleotides downstream from the 3' splice site. The former site occurs just after the sequence GGU, and may indicate the existence of a GGU-binding site within the IVS RNA. The latter site follows the sequence CUCU, which is identical with the four nucleotides preceding the 5' splice site. This observation led to a model where where the CUCU sequence in the 3' exon acts as a cryptic 5' splice site. The model predicted the existence of a circular RNA containing the first 52 nucleotides of the 3' exon. A small circular RNA was isolated and partially sequenced and found to support the model. So, a cryptic 5' splice site can function even if it is located downstream from the 3' splice site. Precursor RNA labeled at its 5' end, presumably by a GTP exchange reaction mediated by the IVS, is also described.
嗜热四膜虫核糖体RNA前体的间隔序列（IVS）在体外能进行精确的自我剪接。本文所展示的工作研究了5'外显子中嗜热四膜虫rRNA序列对于剪接准确性和效率的要求。构建了三种质粒，分别带有天然5'外显子序列的九个、四个和两个核苷酸，随后是IVS以及嗜热四膜虫3'外显子的26个核苷酸。体外从这些质粒转录RNA并测试其自我剪接活性。随着天然5'外显子序列被质粒序列取代，通过连接外显子的产生来衡量的剪接效率降低。即使天然5'外显子序列仅剩下四个核苷酸，精确剪接仍会持续。当天然外显子仅剩下两个核苷酸时，则未观察到连接的外显子。随着正常反应效率降低，新的RNA种类产生的量越来越多。对这些新的RNA种类进行了检测，发现它们是前体RNA异常反应的产物。其中两种异常反应涉及GTP自动添加到3'剪接位点下游六个核苷酸和52个核苷酸处的位点。前一个位点刚好在GGU序列之后，可能表明IVS RNA中存在一个GGU结合位点。后一个位点在CUCU序列之后，该序列与5'剪接位点之前的四个核苷酸相同。这一观察结果导致了一个模型，其中3'外显子中的CUCU序列充当隐蔽的5'剪接位点。该模型预测存在一种包含3'外显子前52个核苷酸的环状RNA。分离出一种小的环状RNA并进行了部分测序，发现其支持该模型。因此，即使隐蔽的5'剪接位点位于3'剪接位点下游，它也能发挥作用。还描述了在其5'端标记的前体RNA，推测是由IVS介导的GTP交换反应所致。
