Processing of Non-Coding RNA by Mlp1 in Tetrahymena thermophila

RNA species are commonly transcribed as precursors and require post-transcriptional processing to become functional mature RNA transcripts. This includes the abundant cytoplasmic transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs) that function in messenger RNA (mRNA) translation. Other non-coding RNAs also require extensive processing and assembly into ribonucleoproteins (RNPs), including small nuclear RNAs (snRNAs) that function in pre-mRNA splicing and small nucleolar RNAs (snoRNAs) involved in the processing and post-transcriptional modification of non-coding RNAs. The ciliate Tetrahymena thermophila is a highly studied eukaryotic model organism, however, many of its RNA processing pathways remain unexplored. In this work, we use molecular biology, biochemistry, cell biology and bioinformatic techniques to investigate the role of a novel La protein, Mlp1, in pre-tRNA, snRNP and snoRNP biogenesis.

Unlike previously studied genuine La proteins, Mlp1 lacks an RNA-binding domain typically required for high-affinity binding of uridylate-tailed La target RNAs. We confirm that Mlp1 performs typical La protein functions, including uridylate-dependent preferential binding of pre-tRNAs and RNA chaperone activity to promote processing and maturation of misfolded nascent pre-tRNAs. However, in contrast to pre-tRNA processing in other eukaryotes, depletion of Mlp1 results in 3′-trailer stabilization instead of rapid trimming by 3′-exonucleases, indicating that Mlp1 is linked to a fundamentally different mechanism of tRNA processing in Tetrahymena thermophila. We also find that Mlp1 associates with mature snRNAs lacking the typical high affinity uridylate binding site and a core protein component of the snRNP. More specifically, Mlp1 interacts with the U4/U6 di-snRNP complex, which is formed during splicing, and Mlp1 depletion affects assembly of this complex. Mlp1 depletion also results in diminished splicing efficiency of pre-mRNAs, further supporting a functional role for Mlp1 in splicing in this system. Lastly, we show that Mlp1 associates with all previously annotated snoRNAs which function as guide RNAs in post-transcriptional modification of non-coding RNAs. We use this as a metric to predict novel snoRNAs in Tetrahymena thermophila and validate expression. Our work demonstrates new roles for the variant genuine La protein Mlp1 in the biogenesis of non-coding RNAs critical for the translation of proteins.