Ahamad Jamaluddin, Ojha Sandeep, Srivastava Ankita, Bhattacharya Alok, Bhattacharya Sudha
School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
Mol Biochem Parasitol. 2015 Jun;201(2):146-52. doi: 10.1016/j.molbiopara.2015.07.006. Epub 2015 Aug 3.
Ribosome synthesis involves all three RNA polymerases which are co-ordinately regulated to produce equimolar amounts of rRNAs and ribosomal proteins (RPs). Unlike model organisms where transcription of rRNA and RP genes slows down during stress, in E. histolytica rDNA transcription continues but pre-rRNA processing slows down and unprocessed pre-rRNA accumulates during serum starvation. To investigate the regulation of RP genes under stress we measured transcription of six selected RP genes from the small- and large-ribosomal subunits (RPS6, RPS3, RPS19, RPL5, RPL26, RPL30) representing the early-, mid-, and late-stages of ribosomal assembly. Transcripts of these genes persisted in growth-stressed cells. Expression of luciferase reporter under the control of two RP genes (RPS19 and RPL30) was studied during serum starvation and upon serum replenishment. Although luciferase transcript levels remained unchanged during starvation, luciferase activity steadily declined to 7.8% and 15% of control cells, respectively. After serum replenishment the activity increased to normal levels, suggesting post-transcriptional regulation of these genes. Mutations in the sequence -2 to -9 upstream of AUG in the RPL30 gene resulted in the phenotype expected of post-transcriptional regulation. Transcription of luciferase reporter was unaffected in this mutant, and luciferase activity did not decline during serum starvation, showing that this sequence is required to repress translation of RPL30 mRNA, and mutations in this region relieve repression. Our data show that during serum starvation E. histolytica blocks ribosome biogenesis post-transcriptionally by inhibiting pre-rRNA processing on the one hand, and the translation of RP mRNAs on the other.
核糖体合成涉及所有三种RNA聚合酶，它们受到协调调控以产生等摩尔量的rRNA和核糖体蛋白（RP）。与模式生物不同，在模式生物中rRNA和RP基因的转录在应激期间会减慢，而在溶组织内阿米巴中，rDNA转录会继续，但前体rRNA加工会减慢，并且在血清饥饿期间未加工的前体rRNA会积累。为了研究应激条件下RP基因的调控，我们测量了来自小核糖体亚基和大核糖体亚基的六个选定RP基因（RPS6、RPS3、RPS19、RPL5、RPL26、RPL30）的转录，这些基因代表核糖体组装的早期、中期和后期阶段。这些基因的转录本在生长应激细胞中持续存在。在血清饥饿期间和血清补充后，研究了两个RP基因（RPS19和RPL30）控制下的荧光素酶报告基因的表达。尽管在饥饿期间荧光素酶转录水平保持不变，但荧光素酶活性分别稳步下降至对照细胞的7.8%和15%。血清补充后，活性增加到正常水平，表明这些基因存在转录后调控。RPL30基因AUG上游-2至-9序列中的突变导致了转录后调控预期的表型。在该突变体中，荧光素酶报告基因的转录不受影响，并且在血清饥饿期间荧光素酶活性没有下降，表明该序列是抑制RPL30 mRNA翻译所必需的，并且该区域的突变可解除抑制。我们的数据表明，在血清饥饿期间，溶组织内阿米巴通过一方面抑制前体rRNA加工，另一方面抑制RP mRNA的翻译，在转录后阻断核糖体生物合成。
