Bcp1、Rpl23及Sqt1形成的小複合體於核醣體生合成的功能研究

Abstract

核醣體生合成是細胞中相當重要的過程，約略有兩百個相關因子參與在此途徑當中。核醣蛋白的生成會在細胞質中，但核醣體的組裝開始於核仁，所以核醣蛋白在組裝前必須先入核。在前人的研究中指出，伴護蛋白或運輸蛋白可以藉由電荷的中和來維持核醣蛋白的穩定性，並且增進其運輸效率。Bcp1已經被證實是PI4P 5-激酶Mss4的運輸蛋白，在本實驗室先前的研究中發現，Bcp1會參與核醣體生合成的途徑，且篩選出了Bcp1突變株的高通量抑制子Rpl23。在本研究中發現，Bcp1會與Rpl23結合，若細胞中缺乏Bcp1時，會造成Rpl23與60S的結合產生問題。另外，Tif6是一個核醣體生合成相關因子，結合在60S上與40S的結合面，防止未成熟的60S進入轉譯步驟，其主要的結合位置就在Rpl23上。當缺乏Bcp1影響到Rpl23時，進而影響到Tif6與60S的結合。然而，當Bcp1突變 (bcp1ts) 時，會滯留於60S上，此時Rpl23雖然於60S上，但亦會使Tif6無法順利結合至60S，影響核醣體的成熟過程。因此，Bcp1應為Tif6結合的一個調控點，當Rpl23被Bcp1協助順利結合至60S後，Bcp1需離開60S，Tif6才能結合至60S上，完成此一步驟。 接著，在本研究中還發現了另一個與Bcp1相關的因子Sqt1，在前人的研究中只知道Sqt1是Rpl10的伴護蛋白，並且主要在細胞質中幫助Rpl10的穩定及和60S的結合。在此我發現Sqt1會與Bcp1及Rpl23有交互作用，並且會進入到細胞核中。在Bcp1及Rpl23的突變株中，會使Sqt1滯留在核醣體上，進而使下游的Nmd3與60S的結合產生問題。另外，改變Sqt1的表現量會影響到Bcp1在細胞中的位置以及含量，顯示Sqt1除了是Rpl10的伴護蛋白外，對於核醣體生合成途徑可能具有其他的功能。

The assembly of a ribosome requires about 200 transacting factors that are generally regulated by growth control and developmental signals. Ribosomal proteins are translated in the cytoplasm and must be imported for further assembly with rRNAs. It has been shown that chaperones or karyopherins could maintain the solubility of ribosomal proteins by neutralizing unfavorable positive charges and thus facilitate the transport. Bcp1 has been identified as a factor necessary for the export of Mss4, a PI4P 5-kinase. Here we have also identified Bcp1 as necessary for the maturation of 60S ribosomal subunits. Cells with mutations of Bcp1 are lethal and have deficient 60S levels. To further characterize its functional role, we conducted a high-copy suppressor screen and identified Rpl23 as a suppressor of bcp1 mutant. Bcp1 is shown to have a physical interaction with Rpl23. Depleting Bcp1 causes deficient loading of Rpl23. In addition, Tif6 loses its nucleolar distribution and 60S interaction. Tif6 is a ribosome biogenesis factor of 60S subunits with predominant nuclear and nucleolar distribution; its homolog in higher eukaryotic cells is eIF6. Tif6 is bound on the 60S subunits, and the C-terminus of Rpl23 is the major interaction site. The binding of Tif6 on the 60S subunits blocks association between 60S and 40S subunits by blocking the inter-subunit bridge formation. This blocking prevents inappropriate interaction with 40S subunits before full maturation of 60S. Interestingly, Rpl23 is loaded properly in bcp1ts mutant, which is persistent on the 60S subunits, and Tif6 is also blocked from binding. We conclude that the retention of Bcp1 causes steric hindrance and prevents Tif6 from assessing Rpl23. We find that Bcp1 is a novel 60S biogenesis transacting factor which works as a chaperone of Rpl23. Once Rpl23 is loaded properly by Bcp1, Tif6 is subsequently bound after Bcp1 is released. We also find another Bcp1-related factor Sqt1, which is previously reported as a chaperone of Rpl10. Sqt1 maintains the stability of Rpl10, and facilitates the loading of Rpl10 onto the 60S in the cytoplasm. Here we find that Sqt1 forms a complex with Bcp1 and Rpl23, and transported into the nucleus together. In Bcp1 or Rpl23 mutant, Sqt1 retends on the ribosome, affecting the loading of Nmd3 downstream. Alterring the expression level of Sqt1 decreases the protein level of Bcp1 and change its cellular localization. These data suggest that Sqt1 has another function involved in the ribosome biogenesis pathway.