Pat1 is a hub for mRNA metabolism, acting in pre-mRNA splicing, translation repression, and mRNA decay. A critical step in all 5′-3′ mRNA decay pathways is removal of the 5′ cap structure, which precedes and permits digestion of the RNA body by conserved exonucleases. During bulk 5′-3′ decay, the Pat1/Lsm1-7 complex engages mRNA at the 3′ end and promotes hydrolysis of the cap structure by Dcp1/Dcp2 at the 5′ end through an unknown mechanism. We reconstitute Pat1 with 5′ and 3′ decay factors and show how it activates multiple steps in late mRNA decay. First, we find that Pat1 stabilizes binding of the Lsm1-7 complex to RNA using two conserved short-linear interaction motifs. Second, Pat1 directly activates decapping by binding elements in the disordered C-terminal extension of Dcp2, alleviating autoinhibition and promoting substrate binding. Our results uncover the molecular mechanism of how separate domains of Pat1 coordinate the assembly and activation of a decapping messenger ribonucleoprotein (mRNP) that promotes 5′-3′ mRNA degradation.

23Pat1 is a hub for mRNA metabolism, acting in pre-mRNA splicing, translation repression 24 and mRNA decay. A critical step in all 5'-3' mRNA decay pathways is removal of the 5' 25 cap structure, which precedes and permits digestion of the RNA body by conserved 26 exonucleases. During bulk 5'-3' decay, the Pat1/Lsm1-7 complex engages mRNA at the 27 3' end and promotes hydrolysis of the cap structure by Dcp1/Dcp2 at the 5' end through 28 an unknown mechanism. We reconstitute Pat1 with 5' and 3' decay factors and show 29how it activates multiple steps in late mRNA decay. First, we find that Pat1 stabilizes 30 binding of the Lsm1-7 complex to RNA using two conserved short-linear interaction 31 motifs. Secondly, Pat1 directly activates decapping by binding elements in the 32 disordered C-terminal extension of Dcp2, alleviating autoinhibition and promoting 33 substrate binding. Our results uncover the molecular mechanism of how separate 34 domains of Pat1 coordinate the assembly and activation of a decapping mRNP that 35 promotes 5'-3' mRNA degradation. 36 37 miRNA mediated decay (4-7). The Dcp1/Dcp2 holoenzyme is a conserved NUDIX 45 hydrolase that cleaves the 5' m 7 G cap and is targeted to specific mRNAs by cofactors 46(1, 8-13). These cofactors regulate the activity of Dcp1/Dcp2 by either binding the 47 catalytic core of the enzyme or helical leucine motifs (HLMs) in the disordered C-48

Pat1 promotes the activation and assembly of multiple proteins during mRNA decay. After deadenylation, the Pat1/Lsm1-7 complex binds to transcripts containing oligo(A) tails, which can be modified by the addition of several terminal uridine residues. Pat1 enhances Lsm1-7 binding to the 3' end, but it is unknown how this interaction is influenced by nucleotide composition. Here we examine Pat1/Lsm1-7 binding to a series of oligoribonucleotides containing different A/U contents using recombinant purified proteins from fission yeast. We observe a positive correlation between fractional uridine content and Lsm1-7 binding affinity. Addition of Pat1 broadens RNA specificity of Lsm1-7 by enhancing binding to A-rich RNAs and increases cooperativity on all oligonucleotides tested. Consistent with increased cooperativity, Pat1 promotes multimerization of the Lsm1-7 complex, which is potentiated by RNA binding. Furthermore, the inherent ability of Pat1 to multimerize drives liquid-liquid phase separation with multivalent decapping enzyme complexes of Dcp1/Dcp2. Our results uncover how Pat1 regulates RNA binding and higher order assembly by mRNA decay factors.

23Pat1 promotes the activation and assembly of multiple proteins during mRNA decay. After 24 deadenylation, the Pat1/Lsm1-7 complex binds to transcripts containing oligo(A) tails, 25 which can be modified by the addition of several terminal uridine residues. Pat1 enhances 26 Lsm1-7 binding to the 3' end, but it is unknown how this interaction is influenced by 27 nucleotide composition. Here we examine Pat1/Lsm1-7 binding to a series of 28 oligoribonucleotides containing different A/U contents using recombinant purified proteins 29 from fission yeast. We observe a positive correlation between fractional uridine content 30 and Lsm1-7 binding affinity. Addition of Pat1 broadens RNA specificity of Lsm1-7 by 31 enhancing binding to A-rich RNAs and increases cooperativity on all oligonucleotides 32 tested. Consistent with increased cooperativity, Pat1 promotes multimerization of the 33 Lsm1-7 complex, which is potentiated by RNA binding. Furthermore, the inherent ability 34 of Pat1 to multimerize drives liquid-liquid phase separation with multivalent decapping 35 enzyme complexes of Dcp1/Dcp2. Our results uncover how Pat1 regulates RNA binding 36 and higher order assembly by mRNA decay factors. 37 38 INTRODUCTION 39