Multilevel Differential Control of Hormone Gene Expression Programs by hnRNP L and LL in Pituitary Cells

Abstract

The pituitary-derived somatolactotrophe GH3 cells secrete both growth hormone (GH) and prolactin (PRL). We have found that the hnRNP L and L-like (LL) paralogs differentially regulate alternative splicing of genes in these cells. Here, we show that hnRNP L is essential for PRL only, but LL is essential for both PRL and GH production. Transcriptome-wide RNA sequencing (RNA-Seq) analysis indicates that they differentially control groups of hormone or hormone-related genes involved in hormone production/regulation at total transcript and alternative exon levels. Interestingly, hnRNP L also specifically binds and prevents the aberrant usage of a nonconserved CA-rich intron piece of Prl pre-mRNA transcripts, and many others involved in endocrine functions, to prevent mostly cryptic last exons and mRNA truncation. Essential for the full hnRNP L effect on specific exons is a proline-rich region that emerged during evolution in vertebrate hnRNP L only but not LL. Together, our data demonstrate that the hnRNP L and its paralog, LL, differentially control hormone gene expression programs at multiple levels, and hnRNP L in particular is critical for protecting the transcriptome from aberrant usage of intronic sequences. The multilevel differential control by hnRNPs likely tailors the transcriptome to help refine and safeguard the different gene expression programs for different hormones.

Transcriptome protection by the expanded family of hnRNPs

“…Moreover, PTBP1 also interacts with Matrin-3 (with two RRMs sharing 38% identity with PTB RRM1) to inhibit the cryptic exons from LINE repeats [32], where the exon usage was increased upon knockdown of both proteins or upon LINE deletion. Similar repression of cryptic exons has also been reported recently for the hnRNP L by two labs independently [25,31]. show that the differentially expanded number of hnRNPs correlates better with the evolving total lengths of genes and genome sizes than the total number of genes among different species.…”

“…The expanded hnRNP family members could be nonredundant in not only RNA binding but also their effects on target exons or genes. For examples, PTBP1 and PTBP2 paralogs could have different target exons though with redundant ones as well in neuronal differentiation [23,24]; similarly, hnRNP L regulates gene expression and alternative splicing involved in hormone production distinctively from its paralog hnRNP LL though with some overlapping targets as well [25]; hnRNP E1 but not its paralog E2 suppresses HIV-1 gene expression [26]. Thus, even the paralogs could have distinct target differences in the regulation of gene expression, let alone the differences among homologues.…”

“…Direct evidence has been obtained for the expanded hnRNPs to inhibit transcriptome-wide cryptic exons of endogenous transcripts in recent years, with large-scale sequencing of RNA samples upon loss-of-function of hnRNPs and analysis of reads mapped to the annotated introns [14,25,[29][30][31][32]. For example, hnRNP C1 knockdown led to global increase of cryptic Alu exon formation and disease-associated deletion in Alu elements disrupted the ability of hnRNP C1 to inhibit the exon formation [14].…”

“…Interestingly, in the case of hnRNP L/LL, there is little overlap of their target cryptic exons between the paralogs [25]. It is mainly hnRNP L that prevents cryptic exon usage.…”

“…To these questions, the following points perhaps could be considered. For relatively simple repetitive sequences in the introns, such as Alu, LINE, UG, CU and CA repeats, a specific set of hnRNPs appear to be sufficient to inhibit cryptic splicing of related intron sequences [14,25,[29][30][31][32]. For more complex sequences, more versatile RNA binding or other properties would be helpful.…”