Subcytoplasmic location of translation controls protein output.

The cytoplasm is highly compartmentalized, but the extent and consequences of subcytoplasmic mRNA localization in non-polarized cells are largely unknown. We determined mRNA enrichment in TIS granules (TGs) and the rough endoplasmic reticulum (ER) through particle sorting and isolated cytosolic mRNAs by digitonin extraction. When focusing on genes that encode non-membrane proteins, we observed that 52% have transcripts enriched in specific compartments. Compartment enrichment correlates with a combinatorial code based on mRNA length, exon length, and 3' UTR-bound RNA-binding proteins. Compartment-biased mRNAs differ in the functional classes of their encoded proteins: TG-enriched mRNAs encode low-abundance proteins with strong enrichment of transcription factors, whereas ER-enriched mRNAs encode large and highly expressed proteins. Compartment localization is an important determinant of mRNA and protein abundance, which is supported by reporter experiments showing that redirecting cytosolic mRNAs to the ER increases their protein expression. In summary, the cytoplasm is functionally compartmentalized by local translation environments.

Extensible benchmarking of methods that identify and quantify polyadenylation sites from RNA-seq data.

The tremendous rate with which data is generated and analysis methods emerge makes it increasingly difficult to keep track of their domain of applicability, assumptions, limitations, and consequently, of the efficacy and precision with which they solve specific tasks. Therefore, there is an increasing need for benchmarks, and for the provision of infrastructure for continuous method evaluation. APAeval is an international community effort, organized by the RNA Society in 2021, to benchmark tools for the identification and quantification of the usage of alternative polyadenylation (APA) sites from short-read, bulk RNA-sequencing (RNA-seq) data. Here, we reviewed 17 tools and benchmarked eight on their ability to perform APA identification and quantification, using a comprehensive set of RNA-seq experiments comprising real, synthetic, and matched 3'-end sequencing data. To support continuous benchmarking, we have incorporated the results into the OpenEBench online platform, which allows for continuous extension of the set of methods, metrics, and challenges. We envisage that our analyses will assist researchers in selecting the appropriate tools for their studies, while the containers and reproducible workflows could easily be deployed and extended to evaluate new methods or data sets.

Quantifying 3'UTR length from scRNA-seq data reveals changes independent of gene expression.

Although more than half of all genes generate transcripts that differ in 3'UTR length, current analysis pipelines only quantify the amount but not the length of mRNA transcripts. 3'UTR length is determined by 3' end cleavage sites (CS). We map CS in more than 200 primary human and mouse cell types and increase CS annotations relative to the GENCODE database by 40%. Approximately half of all CS are used in few cell types, revealing that most genes only have one or two major 3' ends. We incorporate the CS annotations into a computational pipeline, called scUTRquant, for rapid, accurate, and simultaneous quantification of gene and 3'UTR isoform expression from single-cell RNA sequencing (scRNA-seq) data. When applying scUTRquant to data from 474 cell types and 2134 perturbations, we discover extensive 3'UTR length changes across cell types that are as widespread and coordinately regulated as gene expression changes but affect mostly different genes. Our data indicate that mRNA abundance and mRNA length are two largely independent axes of gene regulation that together determine the amount and spatial organization of protein synthesis.

The FXR1 network acts as signaling scaffold for actomyosin remodeling.

It is currently not known that mRNAs fulfill structural roles in the cytoplasm. Here, we report the FXR1 network, an mRNA-protein (mRNP) network present throughout the cytoplasm: FXR1 packages exceptionally long mRNAs that serve as an underlying network scaffold and concentrate FXR1 molecules, which have multiple protein binding sites. The proximity of FXR1 molecules makes the FXR1 network a hub for transient interactions of proteins lacking RNA-binding domains. We show that the FXR1 network is necessary for RhoA signaling-induced actomyosin reorganization to provide spatial proximity between kinases and their substrates. A point mutation in FXR1, which is found in its FMR1 homolog and causes Fragile X syndrome, disrupts the network. FXR1 network disruption prevents actomyosin remodeling-an essential and ubiquitous process for the regulation of cell shape, migration, and synaptic function. These findings uncover a structural role for cytoplasmic mRNA and show how the FXR1 RNA-binding protein as part of the FXR1 network acts as organizer of signaling reactions.

Extensible benchmarking of methods that identify and quantify polyadenylation sites from RNA-seq data.

The tremendous rate with which data is generated and analysis methods emerge makes it increasingly difficult to keep track of their domain of applicability, assumptions, and limitations and consequently, of the efficacy and precision with which they solve specific tasks. Therefore, there is an increasing need for benchmarks, and for the provision of infrastructure for continuous method evaluation. APAeval is an international community effort, organized by the RNA Society in 2021, to benchmark tools for the identification and quantification of the usage of alternative polyadenylation (APA) sites from short-read, bulk RNA-sequencing (RNA-seq) data. Here, we reviewed 17 tools and benchmarked eight on their ability to perform APA identification and quantification, using a comprehensive set of RNA-seq experiments comprising real, synthetic, and matched 3'-end sequencing data. To support continuous benchmarking, we have incorporated the results into the OpenEBench online platform, which allows for seamless extension of the set of methods, metrics, and challenges. We envisage that our analyses will assist researchers in selecting the appropriate tools for their studies. Furthermore, the containers and reproducible workflows generated in the course of this project can be seamlessly deployed and extended in the future to evaluate new methods or datasets.

Enhancers regulate 3' end processing activity to control expression of alternative 3'UTR isoforms.

Multi-UTR genes are widely transcribed and express their alternative 3'UTR isoforms in a cell type-specific manner. As transcriptional enhancers regulate mRNA expression, we investigated if they also regulate 3'UTR isoform expression. Endogenous enhancer deletion of the multi-UTR gene PTEN did not impair transcript production but prevented 3'UTR isoform switching which was recapitulated by silencing of an enhancer-bound transcription factor. In reporter assays, enhancers increase transcript production when paired with single-UTR gene promoters. However, when combined with multi-UTR gene promoters, they change 3'UTR isoform expression by increasing 3' end processing activity of polyadenylation sites. Processing activity of polyadenylation sites is affected by transcription factors, including NF-κB and MYC, transcription elongation factors, chromatin remodelers, and histone acetyltransferases. As endogenous cell type-specific enhancers are associated with genes that increase their short 3'UTRs in a cell type-specific manner, our data suggest that transcriptional enhancers integrate cellular signals to regulate cell type-and condition-specific 3'UTR isoform expression.

Generation of 3'UTR knockout cell lines by CRISPR/Cas9-mediated genome editing.

In addition to the protein code, messenger RNAs (mRNAs) also contain untranslated regions (UTRs). 3'UTRs span the region between the translational stop codon and the poly(A) tail. Sequence elements located in 3'UTRs are essential for pre-mRNA processing. 3'UTRs also contain elements that can regulate protein abundance, localization, and function. At least half of all human genes use alternative cleavage and polyadenylation (APA) to further diversify the regulatory potential of protein functions. Traditional gene editing approaches are designed to disrupt the production of functional proteins. Here, we describe a method that allows investigators to manipulate 3'UTR sequences of endogenous genes for both single- 3'UTR and multi-3'UTR genes. As 3'UTRs can regulate individual functions of proteins, techniques to manipulate 3'UTRs at endogenous gene loci will help to disentangle multi-functionality of proteins. Furthermore, the ability to directly examine the impact of gene regulatory elements in 3'UTRs will provide further insights into their functional significance.

Two high-risk susceptibility loci at 6p25.3 and 14q32.13 for Waldenström macroglobulinemia.

Waldenström macroglobulinemia (WM)/lymphoplasmacytic lymphoma (LPL) is a rare, chronic B-cell lymphoma with high heritability. We conduct a two-stage genome-wide association study of WM/LPL in 530 unrelated cases and 4362 controls of European ancestry and identify two high-risk loci associated with WM/LPL at 6p25.3 (rs116446171, near EXOC2 and IRF4; OR = 21.14, 95% CI: 14.40-31.03, P = 1.36 × 10-54) and 14q32.13 (rs117410836, near TCL1; OR = 4.90, 95% CI: 3.45-6.96, P = 8.75 × 10-19). Both risk alleles are observed at a low frequency among controls (~2-3%) and occur in excess in affected cases within families. In silico data suggest that rs116446171 may have functional importance, and in functional studies, we demonstrate increased reporter transcription and proliferation in cells transduced with the 6p25.3 risk allele. Although further studies are needed to fully elucidate underlying biological mechanisms, together these loci explain 4% of the familial risk and provide insights into genetic susceptibility to this malignancy.