UPSTREAM INTRONS INFLUENCE THE EFFICIENCY OF FINAL INTRON REMOVAL AND RNA 3'-END FORMATION
UPSTREAM INTRONS INFLUENCE THE EFFICIENCY OF FINAL INTRON REMOVAL AND RNA 3'-END FORMATION
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DOI：
10.1101/gad.8.3.363
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发表时间：
1994-02-01
影响因子：
10.5
通讯作者：
MAQUAT, LE
中科院分区：
文献类型：
--
作者：
NESIC, D;MAQUAT, LE
对于使用活细胞或无细胞提取物检测的所有含内含子的高级真核生物的前mrna，在3‘端内含子中需要一个功能性的3’剪接位点才能有效地形成RNA 3'端。内含子序列促进RNA 3'端形成的机制，这是通过核内溶裂解和聚腺苷化实现的，尚不清楚。我们在这里报道，在完整的细胞中，RNA 3‘端形成的效率与最终内含子去除的效率相关，即使内含子通常是5’端或内部内含子。因此，3‘端内含子对3’端形成的影响可能归因于剪接效率的决定因素，其中包括但不限于3'端剪接位点。采用定量RNase定位和反转录与聚合酶链反应相结合的方法来评估人类三磷酸异构酶（TPI）基因中内含子缺失对RNA 3'端形成的影响。结果表明，TPI RNA 3'端的形成需要TPI基因的内含子以及最后的内含子6才能有效地进行。这些额外的TPI基因内含子也是有效去除内含子6所必需的。当内含子1和5被设计成最后的内含子时，发现它们和内含子6一样，在RNA 3'端形成中起作用，其效率与它们的去除效率相关。内含子6的5′和3′剪接位点的同时缺失降低了RNA 3′端形成和内含子5的去除效率，内含子5构成了3′功能最强的内含子。仅删除内含子6的3‘剪接位点可阻止RNA 3’端形成，但对内含子5的去除效率没有影响。仅删除内含子6的5‘剪接位点，导致外显子6跳变（即内含子5、外显子6和内含子6作为一个单元被去除），对RNA 3’端形成的效率或内含子5-外显子6-内含子6的去除都没有影响。这些结果表明，3‘端内含子内的序列通过在最后两个外显子之间形成的相互作用网络，很可能在RNA加工因子之间，与RNA 3’端的形成和倒数第二个内含子的去除在功能上偶联。
For all intron-containing pre-mRNAs of higher eukaryotes that have been examined using either living cells or cell-free extracts, a functional 3' splice site within the 3'-terminal intron is required for efficient RNA 3'-end formation. The mechanism by which intron sequences facilitate RNA 3'-end formation, which is achieved by endonucleolytic cleavage and polyadenylation, is not understood. We report here that in intact cells the efficiency of RNA 3'-end formation correlates with the efficiency of final intron removal, even when the intron is normally a 5'-terminal or internal intron. Therefore, the influence of the 3'-terminal intron on 3'-end formation is likely to be attributable to the determinants of splicing efficiency, which include but are not limited to the 3' splice site. Quantitative RNase mapping and methods that couple reverse transcription and the polymerase chain reaction were used to assess the consequence to RNA 3'-end formation of intron deletions within the human gene for triosephosphate isomerase (TPI). Results indicate that the formation of TPI RNA 3' ends requires TPI gene introns in addition to the last intron, intron 6, to proceed efficiently. These additional TPI gene introns are also required for the efficient removal of intron 6. When introns 1 and 5 were engineered to be the final intron, they were found, as was intron 6, to function in RNA 3'-end formation with an efficiency that correlated with their efficiency of removal. The simultaneous deletion of the 5' and 3' splice sites of intron 6 reduced the efficiencies of both RNA 3'-end formation and the removal of intron 5, which constituted the 3'-most functional intron. Deletion of only the 3' splice site of intron 6 precluded RNA 3'-end formation but had no effect on the efficiency of intron 5 removal. Deletion of only the 5' splice site of intron 6, which resulted in exon 6 skipping (i.e., the removal of intron 5, exon 6, and intron 6 as a single unit), had no effect on the efficiencies of either RNA 3'-end formation or the removal of intron 5-exon 6-intron 6. These results indicate that sequences within the 3'-terminal intron are functionally coupled to both RNA 3'-end formation and removal of the penultimate intron via a network of interactions that form across the last two exons and, most likely, between RNA processing factors.