Studying MTREC complex and its role in nuclear RNA surveillance

Abstract

Eukaryotic genomes are pervasively transcribed. However, these pervasive transcripts such as Cryptic Unstable Transcripts (CUTs) are rapidly degraded by the RNA surveillance machinery. In Schizosaccharomyces pombe, the evolutionarily conserved 11-subunit MTREC complex is responsible for detecting CUTs and aberrant mRNAs and targeting them to the nuclear exosome for degradation. But the mechanism by which MTREC complex is recruited to different subclasses of CUTs is not understood and forms the central question of my thesis. MTREC complex is comprised of the CAP binding complex Cbc1-Cbc2-Ars2 (CBCA), Iss10-Mmi1-Red5-Pab2-Rmn1, canonical poly(A) polymerase Pla1, Mtr4-like protein 1 helicase Mtl1 and zinc-finger protein Red1. For my study, I primarily concentrated on two of the most intriguing subunit proteins of MTREC complex, the helicase Mtl1 and poly(A) polymerase Pla1. I used a combination of in-vitro and in-vivo techniques to understand the role of these subunit proteins in the MTREC complex mediated exosome targeting of CUTs.

In our study we found that Red1 acts as the central scaffolding protein of MTREC and interacts with each submodule, bridging them to the helicase Mtl1, enabling the unwinding of target RNAs, feeding it to the nuclear exosome. To further characterise the role of Mtl1 subunit, point mutations were made disrupting Red1 binding to Mtl1. Studies conducted in Red1-Mtl1 interaction mutants revealed that while the entire MTREC complex is not essential for cell survival, a truncated MTREC complex without Mtl1 is lethal for the cells. A similar strategy was employed to understand the role of Pla1 in the MTREC complex mediated exosome targeting of CUTs. Our in-vitro experiments revealed that Pla1 specifically recognizes a largely unstructured region of Red1. I disrupted the Pla1-Red1 interaction using specific point mutations and in-vivo high throughput sequencing analysis was carried out in the interaction mutants. This revealed a widespread accumulation of PROMPTs, a sub-class of CUTs, hinting that Pla1 in MTREC complex is essential for PROMPT degradation. Furthermore, RNA-immunoprecipitation (RIP) followed by direct RNA sequencing revealed that these PROMPTs harbored shorter poly(A) tails suggesting that the hyperadenylation of PROMPTs is indeed a function of Pla1 in MTREC complex. We wondered what the role of a hyper adenylated tail is, is it possible that there is an interplay between Pla1 and the poly(A) binding protein-Pab2 of the MTREC complex. RIP-followed by sequencing analysis of Pab2 associated RNA transcripts showed that the extra poly(A) leads to an increased Pab2 binding on PROMPTs. ChIP-seq analysis performed to further understand the formation of facultative heterochromatin islands in the Pla1-Red1 interaction mutants also revealed severe reduction of H3K9me2 enrichment in our mutants, hinting at a possibility that the facultative heterochromatin island formation near meiotic genes also occurs via Pla1 in MTREC complex.

Together, our results reveal that both Mtl1 and Pla1 subunit proteins have crucial role in targeting CUTs to exosome. While Mtl1 is essential for cell survival, Pla1 plays critical role in degradation of PROMPTs and in the formation of facultative heterochromatin islands near meiotic genes. Studies with Pab2 also hints at a probable interplay between Pla1 and Pab2 in MTREC mediated exosome targeting of PROMPTs. Hence, it is possible that Pla1 in MTREC has a much critical role than we understand today. Show more