Abstract
The Drosophila integrator complex consists of 14 subunits that associate with the C terminus of Rpb1 and catalyze the endonucleolytic cleavage of nascent snRNAs near their 3' ends. Although disruption of almost any integrator subunit causes snRNA misprocessing, very little is known about the role of the individual subunits or the network of structural and functional interactions that exist within the complex. Here we developed an RNAi rescue assay in Drosophila S2 cells to identify functional domains within integrator subunit 12 (IntS12) required for snRNA 3' end formation. Surprisingly, the defining feature of the Ints12 protein, a highly conserved and centrally located plant homeodomain finger domain, is not required for reporter snRNA 3' end cleavage. Rather, we find a small, 45-amino acid N-terminal microdomain to be both necessary and nearly sufficient for snRNA biogenesis in cells depleted of endogenous IntS12 protein. This IntS12 microdomain can function autonomously, restoring full integrator processing activity when introduced into a heterologous protein. Moreover, mutations within the microdomain not only disrupt IntS12 function but also abolish binding to other integrator subunits. Finally, the IntS12 microdomain is sufficient to interact and stabilize the putative scaffold integrator subunit, IntS1. Collectively, these results identify an unexpected interaction between the largest and smallest integrator subunits that is essential for the 3' end formation of Drosophila snRNA.
Highlights
Small nuclear RNA 3Ј end processing is carried out by the poorly understood integrator complex
To confirm that dsRNA depletion of integrator subunit 12 (IntS12) led to misprocessing of endogenous Small nuclear RNA (snRNA), we isolated total RNA and performed quantitative real-time PCR analysis using primers that detect the misprocessed forms of the U1, U2, U4, U5, and U6 snRNAs (Fig. 1C)
IntS12 protein expressed from Mt3- or Mt5-containing constructs poorly associated with endogenous IntS1 or IntS9, and this association was completely absent in cells expressing the ⌬N IntS12 microdomain truncation (Fig. 5C, lanes 6, 8, and 10 versus lane 4)
Summary
Small nuclear RNA (snRNA) 3Ј end processing is carried out by the poorly understood integrator complex. We find a small, 45-amino acid N-terminal microdomain to be both necessary and nearly sufficient for snRNA biogenesis in cells depleted of endogenous IntS12 protein This IntS12 microdomain can function autonomously, restoring full integrator processing activity when introduced into a heterologous protein. The only well established protein-protein interaction among integrator subunits is between IntS9 and IntS11 [14, 15] These two proteins contain highly conserved metallo--lactamase and -CASP (CPSF, Artemis, SNM1/PSO2) domains and likely represent the catalytic core of the complex (reviewed in Ref. 16). PHD fingers possess specificity toward unique chemical modifications of amino acids in histone H3 with a particular preference toward lysine methylation These attributes make analysis of the role of the IntS12 PHD finger in snRNA 3Ј end formation an attractive entry point to further our understanding of integrator subunit function. These results suggest that a critical regulatory function for IntS1 and IntS12 is required for integrator activity in snRNA 3Ј end formation
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
