Identification of diverse target RNAs that are functionally regulated by human Pumilio proteins

Abstract

Human Pumilio proteins, PUM1 and PUM2, are sequence specific RNA-binding proteins that regulate protein expression. We used RNA-seq, rigorous statistical testing and an experimentally derived fold change cut-off to identify nearly 1000 target RNAs—including mRNAs and non-coding RNAs—that are functionally regulated by PUMs. Bioinformatic analysis defined a PUM Response Element (PRE) that was significantly enriched in transcripts that increased in abundance and matches the PUM RNA-binding consensus. We created a computational model that incorporates PRE position and frequency within an RNA relative to the magnitude of regulation. The model reveals significant correlation of PUM regulation with PREs in 3′ untranslated regions (UTRs), coding sequences and non-coding RNAs, but not 5′ UTRs. To define direct, high confidence PUM targets, we cross-referenced PUM-regulated RNAs with all PRE-containing RNAs and experimentally defined PUM-bound RNAs. The results define nearly 300 direct targets that include both PUM-repressed and, surprisingly, PUM-activated target RNAs. Annotation enrichment analysis reveal that PUMs regulate genes from multiple signaling pathways and developmental and neurological processes. Moreover, PUM target mRNAs impinge on human disease genes linked to cancer, neurological disorders and cardiovascular disease. These discoveries pave the way for determining how the PUM-dependent regulatory network impacts biological functions and disease states.

Human PUM1 and PUM2 exhibit regulation of divergent mRNA targets in male germ cells

“…It is important to note that several of our RIP-Seq identified targets overlapped with mRNAs previously identified in HeLa (Galgano et al, 2008) and HEK293 (Bohn et al, 2018, Hafner et al, 2010 cells, validating our results. However, it is also important to bear in mind that PUM-mediated activation or repression, or lack of PUM regulation may be cell-typespecific (Cottrell, Chaudhari et al, 2018).…”

“…1D). These numbers are in agreement with those obtained from HeLa cells (Galgano et al, 2008) as well as recent data from HEK293T cells (Bohn et al, 2018).…”

“…Our result showing that PUM1 and PUM2 share only ~10% of their mRNA targets ( Fig. 1H) was surprising given that PUM1 and PUM2 recognize the same UGUAHAUW motif (Bohn et al, 2018) and show remarkably high similarity in binding potential across 12,285 sequences, as shown by quantitative analysis of RNA on a massively parallel array (RNA-MaP) (Jarmoskaite, Denny et al, 2019). Since N-terminal regions are known to be structurally divergent (Spassov & Jurecic, 2003), they might function differently in PUM1 and PUM2, for example, by binding different sets of protein cofactors.…”

“…Although it was previously established that each human PUM paralogue specifically recognizes the PBE motif UGUANAUA (Galgano et al, 2008), the PBE motif UGUAHAUW (H stands for A, C or U while W for A or U) was found in a recent study to be more accurate for PUM1 and PUM2 (Bohn, Van Etten et al, 2018). Therefore, in this study, mRNAs identified by RIP-Seq were screened for the presence of the UGUAHAUW motif.…”

“…Since mRNA binding alone does not imply regulation by RBPs, and PUM1-repressed mRNAs have been reported to undergo degradation (Morris, Mukherjee et al, 2008) or activation (Bohn et al, 2018), we next sought to identify those mRNAs whose expression was modified upon PUM1 or PUM2 gene knockdown. At 72 h after TCam-2 cell transfection with PUM1 or PUM2 siRNA, when silencing efficiency was the highest for both paralogues (Fig.…”