Autoantigen La Promotes Efficient RNAi, Antiviral Response, and Transposon Silencing by Facilitating Multiple-Turnover RISC Catalysis

Abstract

SUMMARY The effector of RNA interference (RNAi) is the RNA-induced silencing complex (RISC). C3PO promotes the activation of RISC by degrading Argonaute2 (Ago2)-nicked passenger strand of duplex siRNA. Active RISC is a multiple-turnover enzyme that uses the guide strand of siRNA to direct Ago2-mediated sequence-specific cleavage of complementary mRNA. How this effector step of RNAi is regulated is currently unknown. Here, we used human Ago2 minimal RISC system to purify Sjögren’s syndrome antigen B (SSB)/autoantigen La as an activator of the RISC-mediated mRNA cleavage activity. Our reconstitution studies showed that La could promote multiple-turnover RISC catalysis by facilitating the release of cleaved mRNA from RISC. Moreover, we demonstrated that La was required for efficient RNAi, antiviral defense, and transposon silencing in vivo. Taken together, the findings of C3PO and La reveal a general concept that regulatory factors are required to remove Ago2-cleaved products to assemble or restore active RISC.

Targeted Deletion of the Gene Encoding the La Autoantigen (Sjögren's Syndrome Antigen B) in B Cells or the Frontal Brain Causes Extensive Tissue Loss

“…As noted in the introduction, mammalian La has been implicated in the regulation of multiple mRNAs, including those for housekeeping functions, such as ribosomal proteins. In addition, La has been implicated in the biogenesis of rRNA and microRNAs (miRNAs) (59)(60)(61). Thus, although we have demonstrated altered pre-tRNA metabolism in the La knockout brain, present technology does not allow us to determine if deficiency in this pathway alone is sufficient to cause cell lethality and the other phenotypes observed.…”

“…Such a function may involve an activity in addition to the binding and protection of pre-tRNAs and/or other RNA polymerase III transcripts that localizes to RRM2. RNA chaperone and other activities that appear to rely on RNA binding distinguishable from UUU-3=-OH binding may be involved (31,62), affecting mRNA translation, as noted above, or an miRNA-related function (60,61). Alternatively, an additional RRM may allow more control or regulation over the pre-RNA or other polymerase III transcript binding activity, perhaps related to the pre-tRNA chaperone activity of RRM1 (17,63).…”

Multiple sensors ensure guide strand selection in human RNAi pathways

“…ssRNAs to be used for siRNA and miRNAs were 5 ′ -phosphorylated. All target RNAs were 41 nt long and were perfectly complementary to their respective guide strands except for mismatches with the last four bases at the 3 ′ end of the guide strand to promote turnover Liu et al 2011). All ssRNAs were gel-purified on a 15% denaturing PAGE gel.…”

The RNA chaperon activity of the human La protein (LARP3)

“…The finding that the RNA chaperone activity of La is required to stimulate both IRES-as well as cap-dependent translation raises the question about the location and the nature of RNA elements/structures recognized by La. It is possible that the chaperone activity of La has an indirect effect on translation by contributing to the processing of RNA molecules required for protein synthesis such as pre-tRNAs [6,7,45] or due to effects on microRNA processing [9,10] . However, 35 S-metabolic-labeling and 2D gel electrophoresis analysis does not suggest that La depletion has a global effect on protein synthesis in HeLa cells [17] .…”

“…La interacts with the precursors of a variety of different RNA molecules e.g. tRNAs, mircoRNAs, snoRNAs [6][7][8] [ [9][10][11] and binds to long viral and cellular mRNAs. Besides its LAM:RRM1-mediated interaction with the oligo(U) 3'-termini common to nascent RNA polymerase III transcripts such as pre-tRNAs [12] , human La can bind to the 5'-terminus of mRNAs referred to as terminal polypyrimidine tract (TOP) mRNAs [13][14][15] , and internal RNA elements in RNAs such as MDM2 [16] , cyclin D1 [17] , XIAP [18] , human immunodeficiency virus [19] , hepatitis C [20] and A virus [21] and polio virus [22] .…”