Anusree Sivadas and Komal Soni have contributed to this work equally.
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. However, the mechanism by which MTREC complex is recruited to different subclasses of CUTs is not understood and forms the central question of my work.
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 this study we primarily concentrated on one of the most intriguing subunit proteins of MTREC complex, the poly(A) polymerase Pla1. We used a combination of in-vitro and in-vivo techniques to understand the role of this subunit protein in the MTREC complex mediated exosome targeting of CUTs.
Our in-vitro experiments using yeast-2-hybrid, ITC, and NMR spectroscopy revealed that Pla1 specifically recognizes a largely unstructured region of Red1. We 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 and ChIP seq analysis performed in the Pla1- Red1 interaction mutants revealed that Pla1 has critical role in degradation of PROMPTs and in the formation of facultative heterochromatin islands near meiotic genes. Studies with Pab2 also hint at a probable interplay between Pla1 and Pab2 in MTREC mediated exosome targeting of CUTs/PROMPTs. Hence, it is possible that Pla1 in MTREC has a more critical role than we understand today.