RNA-binding Protein Lin28 Research Articles

LIN28-Targeting Chromenopyrazoles and Tetrahydroquinolines Induced Cellular Morphological Changes and Showed High Biosimilarity with BRD PROTACs.

The probing of small molecules with heterocyclic scaffolds covering unexplored chemical space and the evaluation of their biological relevance are essential parts of forward chemical genetics approaches and for the development of potential small-molecule therapeutics. In this study, we profiled sets of chromenopyrazoles (CMPs) and tetrahydroquinolines (THQs), originally developed to target the protein-RNA interaction of LIN28-let-7, in a cell painting assay (CPA) measuring cellular morphological changes. Selected LIN28-inactive CMPs and THQs induced cellular morphological changes to different extents. The most CPA-active CMPs 2 and 3 exhibited high bio-similarity with the LCH and BET clusters, while the most CPA-active THQs 13 and 20 indicated a mechanism of action beyond the currently established biosimilarity clusters. Overall, this work demonstrated that CPA is useful in revealing "hidden" biological targets and mechanisms of action for biologically inactive small molecules, which are CMPs and THQs targeting the RNA-binding protein LIN28 in this case, evaluated in target-based strategies. When compared with annotated reference compounds, CMP 3 exhibited a high biosimilarity with the dual BRD7/9 degrading PROTAC VZ185, suggesting that CPA could potentially function as a new phenotypic approach to identify degrader molecules.

Lin28 affects the proliferation and osteogenic differentiation of human dental pulp stem cells by directly inhibiting let-7b maturation

ObjectiveActivation of Lin28 gene under certain conditions promotes tissue damage repair. However, it remains unknown whether conditional expression of Lin28 facilitates the recovery of damaged pulp tissue. In the study, we focus on exploring the effects and possible regulatory mechanisms of Lin28 on the proliferation and differentiation of human dental pulp stem cells (hDPSCs).Materials and methodsWe adopted techniques such as the ethynyl-2ʹ-deoxyuridine (EdU) incorporation assay, RNA-protein immunoprecipitation (RIP) analysis, and luciferase assays to study the regulation of hDPSCs by Lin28. Furthermore, gain-of-function and loss-of-function analyses were also used in explored factors regulating hDPSCs activation.ResultsThe results show that Lin28 inhibited osteogenic differentiation by directly targets pre-let-7b. Through bioinformatics sequencing and dual luciferase experiments we learned that let-7b directly targets the IGF2BP2 3’UTR. Silencing of IGF2BP2 showed a similar biological effect as overexpression of let-7b. Overexpression of IGF2BP2 counteracted the differentiation-promoting effects produced by let-7b overexpression.Discussion/conclusionsIn conclusion, the RNA-binding protein Lin28 regulates osteogenic differentiation of hDPSCs by inhibiting let-7 miRNA maturation. And mature let-7b directly regulated the expression of IGF2BP2 by targeting the 3’UTR region of IGF2BP2 mRNA thus further inhibiting the differentiation of hDPSCs.

Dynamics of miRNA accumulation during C. elegans larval development.

Temporally and spatially controlled accumulation underlies the functions of microRNAs (miRNAs) in various developmental processes. In Caenorhabditis elegans, this is exemplified by the temporal patterning miRNAs lin-4 and let-7, but for most miRNAs, developmental expression patterns remain poorly resolved. Indeed, experimentally observed long half-lives may constrain possible dynamics. Here, we profile miRNA expression throughout C. elegans postembryonic development at high temporal resolution, which identifies dynamically expressed miRNAs. We use mathematical models to explore the underlying mechanisms. For let-7, we can explain, and experimentally confirm, a striking stepwise accumulation pattern through a combination of rhythmic transcription and stage-specific regulation of precursor processing by the RNA-binding protein LIN-28. By contrast, the dynamics of several other miRNAs cannot be explained by regulation of production rates alone. Specifically, we show that a combination of oscillatory transcription and rhythmic decay drive rhythmic accumulation of miR-235, orthologous to miR-92 in other animals. We demonstrate that decay of miR-235 and additional miRNAs depends on EBAX-1, previously implicated in target-directed miRNA degradation (TDMD). Taken together, our results provide insight into dynamic miRNA decay and establish a resource to studying both the developmental functions of, and the regulatory mechanisms acting on, miRNAs.

N-Biphenyl Pyrrolinones and Dibenzofurans as RNA-Binding Protein LIN28 Inhibitors Disrupting the LIN28-Let-7 Interaction.

The RNA-binding protein LIN28 is a regulator of miRNA let-7 biogenesis. Inhibitors of LIN28 are highly sought after given the central role that LIN28 plays in tumorigenesis and development of cancer stem cells as well as LIN28's association with poor clinical prognosis. Although LIN28 inhibitors of different scaffolds have been reported, the potential of most LIN28 inhibiting small molecules was not fully explored since very limited structure-activity relationship (SAR) studies have been performed. We previously identified trisubstituted pyrrolinones as a new class of LIN28 inhibitors disrupting the LIN28-let-7 interaction. Here, we performed extensive SAR by evaluating 95 small molecules and identified new trisubstituted pyrrolinones featuring either an N-biphenyl or N-dibenzofuran substituent, overthrowing the existing conclusion that a salicylic acid moiety is indispensable for activity. Exchange of the negatively charged salicylic acid moiety in LIN28 inhibitors with a heterocyclic substituent is beneficial for membrane permeability, leading to increased activity in a cellular assay, and will potentially reduce toxicity.

Spirocyclic Chromenopyrazole Inhibitors Disrupting the Interaction between the RNA-binding protein LIN28 and Let-7.

Small-molecule inhibitors of the RNA-binding and regulating protein LIN28 have the potential to be developed as chemical probes for biological perturbation and as therapeutic candidates. Reported small molecules disrupting the interaction between LIN28 and let-7 miRNA suffer from moderate to weak inhibitory activity and flat structure-activity relationship, which hindered the development of next-generation LIN28 inhibitors that warrant further evaluations. We report herein the identification of new LIN28 inhibitors utilizing a spirocyclization strategy based on a chromenopyrazole scaffold. Representative compounds 2-5 showed potent in vitro inhibitory activity against LIN28-let-7 interaction and single-digit micromolar potency in inhibiting the proliferation of LIN28-expressed JAR cancer cells. The spirocyclic compound 5 incorporated a position that is amenable for functional group appendage and further structural modifications. The binding mode of compound 5 with the LIN28 cold shock domain was rationalized via a molecular docking analysis.

Lin-28 Homolog B-Activated Protein Disulfide Isomerase A4 Regulates Cell Proliferation, Migration and Invasion of Glioma

The goal of this study is to elucidate the role of protein disulfide isomerase A4 (PDIA4) in glioma, as well as its regulatory mechanism. Cell transfection was performed to adjust the expression level of PDIA4 and RNA-binding protein lin-28 homolog B (LIN28B). The expression of PDIA4 in human astrocytes and glioma cell lines was determined by quantitative real-time PCR and western blot. CCK-8, colony formation, Transwell and wound-healing assays were applied to determine the capabilities of cells to proliferate, invade and migrate. The connection between PDIA4 and LIN28B was demonstrated by RNA immunoprecipitation (RIP) and RNA pull down assays. As a result, PDIA4 was elevated in glioma. PDIA4 depletion hugely suppressed cell proliferative ability, which was characterized by the reduced cell viability and colony formation, and declined contents of PCNA and Ki67. Meanwhile, PDIA4 knockdown repressed the cell capabilities to migrate and invade, accompanied with downregulated MMP2 and MMP9. LIN28N was also found to be upregulated in glioma cells, and was verified to bind with PDIA4 and positively regulate PDIA4 expression. Additionally, LIN28B overexpression partly hindered the suppressive impacts of PDIA4 knockdown on cell abilities to proliferate, migrate and invade. In conclusion, this study delineates that LIN28B-mediated PDIA4 plays a critical role in the progression of glioma.

RNA-binding protein complex LIN28/MSI2 enhances cancer stem cell-like properties by modulating Hippo-YAP1 signaling and independently of Let-7

The RNA binding protein LIN28 directly modulates the stability and translation of target mRNAs independently of Let-7; however, the key downstream targets of LIN28 in this process are largely unknown. Here, we revealed that Hippo signaling effector YAP1 functioned as a key downstream regulator of LIN28 to modulate the cancer stem cell (CSC)-like properties and tumor progressions in triple negative breast cancer (TNBC). LIN28 was overexpressed in BC tissues and cell lines, and significantly correlated with poorer overall survivals in patients. Ectopic LIN28 expression enhanced, while knockdown of LIN28A inhibited the CSC-like properties, cell growth and invasive phenotypes of TNBC cells in vitro and in vivo. Transcriptome analysis demonstrated LIN28 overexpression significantly induced the expressions of YAP1 downstream genes, while reduced the transcripts of YAP1 upstream kinases, such as MST1/2 and LATS1/2, and knockdown of LIN28A exhibited the opposite effects. Furthermore, constitutive activation of YAP1 in LIN28 knockdown TNBC cells could rescue the cell growth and invasive phenotypes in vitro and in vivo. Mechanistically, instead of the dependence of Let-7, LIN28 recruited RNA binding protein MSI2 in a manner dependent on the LIN28 CSD domain and MSI2 RRM domain, to directly induce the mRNA decay of YAP1 upstream kinases, leading to the inhibition of Hippo pathway and activation of YAP1, which eventually gave rise to increased CSC populations, enhanced tumor cell growth and invasive phenotypes. Accordingly, co-upregulations of LIN28 and MSI2 in TNBC tissues were strongly associated with YAP1 protein level and tumor malignance. Taken together, our findings unravel a novel LIN28/MSI2-YAP1 regulatory axis to induce the CSC-like properties, tumor growth and metastasis, independently of Let-7, which may serve as a potential therapeutic strategy for the treatment of a subset of TNBC with LIN28 overexpression.

Mechanism of RNA-binding protein Lin28 in neuronal ferroptosis after intracerebral haemorrhage.

Intracerebral haemorrhage (ICH) is a highly risky cerebrovascular disease with poor prognosis. Lin-28 homolog A (Lin28) has been identified as a crucial regulator in ICH. This study aims to analyse the mechanism of Lin28 in neuronal ferroptosis after ICH and provide theoretical basis for ICH treatment. An ICH mouse model was established via injection of collagenase VII, followed by neurological impairment assessment, and haematoxylin-eosin staining. An in vitro ICH model was established using hemin treatment. Next, cell viability and ferroptosis parameters were detected via cell counting kit-8, assay kits, enzyme-linked immunosorbent assay and western blot. Lin28 expression and tripartite motif-containing 37 (Trim37) mRNA level were detected via western blot and quantitative real-time polymerase chain reaction (qRT-PCR). The binding relationship of Lin28 and Trim37 was verified. ICH mice exhibited neuronal ferroptosis and upregulation of Lin28. Lin28 inhibition alleviated neurological impairment, manifested by decreased hematoma, oedema, neuronal necrosis, glial cell swelling, intracellular vacuoles and inflammatory cell infiltration, reduced Fe2+ concentration and reactive oxygen species content, and increased glutathione and glutathione peroxidase 4 activity. In the hemin-induced HT-22 cells, Lin28 inhibition promoted cell viability and alleviated neuronal ferroptosis. Lin28 bound to Trim37 mRNA to stabilize the mRNA level of Trim37. Overexpression of Trim37 reversed the alleviating role of silencing Lin28 in neuronal ferroptosis after ICH. Overall, Lin28 stabilized the mRNA level of Trim37 to aggravate neuronal ferroptosis after ICH.

Small molecules with tetrahydroquinoline-containing Povarov scaffolds as inhibitors disrupting the Protein–RNA interaction of LIN28–let-7

Inhibition of the RNA-binding protein LIN28 and disruption of the protein–RNA interaction of LIN28–let-7 with small molecules holds great potential to develop new anticancer therapeutics. Herein, we report the LIN28 inhibitory activities of a series of 30 small molecules with a tricyclic tetrahydroquinoline (THQ)-containing scaffold obtained from a Povarov reaction. The THQ molecules were structurally optimized by varying the 2-benzoic acid substituent, the fused ring at 3- and 4-positions, and the substituents at the phenyl moiety of the tetrahydroquinoline core. Among the tested compounds, GG-43 showed dose-dependent inhibition in an EMSA validation assay and low micromolar inhibitory activity in a fluorescence polarization-based assay measuring disruption of LIN28–let-7 interaction. Binding mode between GG-43 and the cold shock domain of LIN28 was proposed via a molecular docking analysis. The study provides one of the first systematic analyses on structural features that are required for LIN28 inhibition, and indicates the necessity to develop small molecules with new scaffolds as LIN28-targeting probes and therapeutic candidates. In parallel, this study demonstrates the polypharmacological nature of tricyclic THQ-containing scaffolds accessible through Povarov reactions.

LINC01094 promotes pancreatic cancer progression by sponging miR-577 to regulate LIN28B expression and the PI3K/AKT pathway

The leading cause of death in pancreatic cancer (PC) patients is the progression of cancer metastasis. Recently, long non-coding RNAs (lncRNAs) have been shown to play an important role in regulating cancer cell proliferation and metastasis; however, its molecular basis in PC remains to be explored. In this study, we observed that LINC01094 was markedly overexpressed in PC tissues and was associated with poor patient prognosis. Downregulation of LINC01094 decreased the proliferation and metastasis of PC cells and inhibited tumorigenesis and metastasis in mouse xenografts. Mechanically, LINC01094 acted as an endogenous miR-577 sponge to increase the expression of its target gene, the RNA-binding protein lin-28 homolog B (LIN28B), by decoying the miR-577, thereby activating the PI3K/AKT pathway. Our findings suggest that LINC01094 plays critical roles in proliferation and metastasis of PC, implying that LINC01094 can be regarded as a new biomarker or therapeutic target for the treatment of PC.

Lin28, a major translation reprogramming factor, gains access to YB-1-packaged mRNA through its cold-shock domain

The RNA-binding protein Lin28 (Lin28a) is an important pluripotency factor that reprograms translation and promotes cancer progression. Although Lin28 blocks let-7 microRNA maturation, Lin28 also binds to a large set of cytoplasmic mRNAs directly. However, how Lin28 regulates the processing of many mRNAs to reprogram global translation remains unknown. We show here, using a structural and cellular approach, a mixing of Lin28 with YB-1 (YBX1) in the presence of mRNA owing to their cold-shock domain, a conserved β-barrel structure that binds to ssRNA cooperatively. In contrast, the other RNA binding-proteins without cold-shock domains tested, HuR, G3BP-1, FUS and LARP-6, did not mix with YB-1. Given that YB-1 is the core component of dormant mRNPs, a model in which Lin28 gains access to mRNPs through its co-association with YB-1 to mRNA may provide a means for Lin28 to reprogram translation. We anticipate that the translational plasticity provided by mRNPs may contribute to Lin28 functions in development and adaptation of cancer cells to an adverse environment.

Lin28 Inhibits the Differentiation from Mouse Embryonic Stem Cells to Glial Lineage Cells through Upregulation of Yap1.

The RNA-binding protein Lin28 regulates neurogliogenesis in mammals, independently of the let-7 microRNA. However, the detailed regulatory mechanism remains obscured. Here, we established Lin28a or Lin28b overexpression mouse embryonic stem cells (ESCs) and found that these cells expressed similar levels of the core pluripotent factors, such as Oct4 and Sox2, and increased Yap1 but decreased lineage-specific markers compared to the control ESCs. Further differentiation of these ESCs to neuronal and glial lineage cells revealed that Lin28a/b overexpression did not affect the expression of neuronal marker βIII-tubulin, but dramatically inhibited the glial lineage markers, such as Gfap and Mbp. Interestingly, overexpression of Yap1 in mouse ESCs phenocopied Lin28a/b overexpression ESCs by showing defect in glial cell differentiation. Inhibition of Yap1/Tead-mediated transcription with verteporfin partially rescued the differentiation defect of Lin28a/b overexpression ESCs. Mechanistically, we demonstrated that Lin28 can directly bind to Yap1 mRNA, and the induction of Yap1 by Lin28a in mESCs is independent of Let7. Taken together, our results unravel a novel Lin28-Yap1 regulatory axis during mESC to glial lineage cell differentiation, which may shed light on glial cell generation in vitro.

Long Non-Coding RNA LINC00355 Promotes the Development and Progression of Colorectal Cancer by Elevating Guanine Nucleotide Exchange Factor T Expression via RNA Binding Protein lin-28 Homolog A.

BackgroundOur previous study showed that guanine nucleotide exchange factor T (GEFT) was highly expressed in colorectal cancer (CRC) tissues and CRC patients with high GEFT expression had a poor prognosis, and suggested the close link of GEFT expression and CRC tumorigenesis/metastasis. In this text, the roles and upstream regulatory mechanisms of GEFT in the development and progression of CRC were further investigated.MethodsExpression levels of GEFT mRNA and LINC00355 was measured by RT-qPCR assay. Protein levels of lin-28 homologue A (LIN28A) and GEFT were determined by western blot assay. Cell proliferative, migratory, and invasive capacities were assessed by CCK-8, Transwell migration and invasion assays, respectively. The effect of GEFT knockdown on CRC tumorigenesis was examined by mouse xenograft experiments in vivo. GEFT mRNA stability was examined by actinomycin D assay. The relationships of LINC000355, LIN28A, and GEFT were explored by RNA pull down and RIP assays.ResultsGEFT was highly expressed in CRC tissues and cell lines. GEFT knockdown inhibited CRC cell proliferation, migration, and invasion, and hindered CRC xenograft tumor growth. GEFT overexpression alleviated the detrimental effects of LINC00355 loss on CRC cell proliferation, migration, and invasion. LINC00355 promoted GEFT expression and enhanced GEFT mRNA stability via LIN28A. LIN28A knockdown weakened the promotive effect of LINC00355 on CRC cell proliferation, migration, and invasion.ConclusionLINC00355 facilitated CRC tumorigenesis and progression by increasing GEFT expression via LIN28A, deepening our understanding on roles and upstream regulatory mechanisms of GEFT in CRC development and progression.

A branched heterochronic pathway directs juvenile-to-adult transition through two LIN-29 isoforms.

Robust organismal development relies on temporal coordination of disparate physiological processes. In Caenorhabditis elegans, the heterochronic pathway controls a timely juvenile-to-adult (J/A) transition. This regulatory cascade of conserved proteins and small RNAs culminates in accumulation of the transcription factor LIN-29, which triggers coordinated execution of transition events. We report that two LIN-29 isoforms fulfill distinct functions. Functional specialization is a consequence of distinct isoform expression patterns, not protein sequence, and we propose that distinct LIN-29 dose sensitivities of the individual J/A transition events help to ensure their temporal ordering. We demonstrate that unique isoform expression patterns are generated by the activities of LIN-41 for lin-29a, and of HBL-1 for lin-29b, whereas the RNA-binding protein LIN-28 coordinates LIN-29 isoform activity, in part by regulating both hbl-1 and lin-41. Our findings reveal that coordinated transition from juvenile to adult involves branching of a linear pathway to achieve timely control of multiple events.

LIN28A loss of function is associated with Parkinson's disease pathogenesis.

Parkinson's disease (PD) is neurodegenerative movement disorder characterized by degeneration of midbrain-type dopamine (mDA) neurons in the substantia nigra (SN). The RNA-binding protein Lin28 plays a role in neuronal stem cell development and neuronal differentiation. In this study, we reveal that Lin28 conditional knockout (cKO) mice show degeneration of mDA neurons in the SN, as well as PD-related behavioral deficits. We identify a loss-of-function variant of LIN28A (R192G substitution) in two early-onset PD patients. Using an isogenic human embryonic stem cell (hESC)/human induced pluripotent stem cell (hiPSC)-based disease model, we find that the Lin28 R192G variant leads to developmental defects and PD-related phenotypes in mDA neuronal cells that can be rescued by expression of wild-type Lin28A. Cell transplantation experiments in PD model rats show that correction of the LIN28A variant in the donor patient (pt)-hiPSCs leads to improved behavioral phenotypes. Our data link LIN28A to PD pathogenesis and suggest future personalized medicine targeting this variant in patients.

LIN28B/IRS1 axis is targeted by miR-30a-5p and promotes tumor growth in colorectal cancer.

Insulin receptor substrate 1 (IRS1) is a potential oncogene that has been implicated in several malignant tumors. However, the regulatory mechanism of IRS1 remains to be investigated. The aim of our current study is to unveil the mechanism by which IRS1 exerts functions in tumorigenesis of colorectal cancer (CRC). The expression level of IRS1 was found to be higher in CRC cells in comparison with the normal cell. To determine the role of IRS1 in regulating CRC cellular processes, loss-of-function assays were designed and carried out in two CRC cell lines. Both in vitro and in vivo functional assays indicated that silencing of IRS1 suppressed CRC cell survival. Based on bioinformatics prediction and mechanism experiments, IRS1 was identified as a downstream target of miR-30a-5p. Furthermore, RNA-binding protein lin-28 homolog B (LIN28B) was determined to be a stabilizer of IRS1 messenger RNA. More importantly, LIN28B also acted as a target of miR-30a-5p.Through rescue assays, we proved that LIN28B-stablized IRS1 mediated miR-30a-5p-mediated CRC cell growth. In conclusion, this study revealed that LIN28B and LIN28B-stablized IRS1 promoted CRC cell growth by cooperating with miR-30a-5p.

MiR-152/LIN28B axis modulates high-glucose-induced angiogenesis in human retinal endothelial cells via VEGF signaling.

Diabetic retinopathy (DR) is a serious complication of diabetes contributing to blindness in patients. Inhibiting retinal neovascularization is a potent strategy for diabetic retinopathy treatment. Reportedly, the stable expression of lin-28 homolog B (LIN28B), a member of the highly conserved RNA-binding proteinLIN28 family, could promote vascular endothelial growth factor (VEGF) expression; herein, we investigated the role and mechanism of LIN28B in diabetic retinopathy progression from the perspective of microRNA (miRNA) regulation. We identified miR-152 as a miRNA that may target the LIN28B 3'-untranslated region and can be significantly downregulated under high-glucose (HG) condition. The expression of miR-152 was remarkably suppressed, whereas the expression of LIN28B was significantly increased under HG condition within both human retinal endothelial cells(hRECs) and retinal microvascular endothelial cell line (hRMECs). miR-152 overexpression significantly suppressed, while LIN28B overexpression promoted the angiogenesis and the protein levels of proangiogenesis factors in both hRECs and hRMECs. More importantly, LIN28B overexpression could remarkably attenuate the effect of miR-152 overexpression. In summary, miR-152 overexpression could inhibit HG-induced angiogenesis in both hRECs and hRMECs via targeting LIN28B and suppressing VEGF signaling. Further, in vivo experiments are needed for the application of miR-152/LIN28B axis in the treatment for diabetic retinopathy.

DLL3 is regulated by LIN28B and miR-518d-5p and regulates cell proliferation, migration and chemotherapy response in advanced small cell lung cancer

Delta-like protein 3 (DLL3) has been reported as a biomarker in various human tumors. However, the biological function and mechanism of it in advanced small cell lung cancer (SCLC) is rarely reported. This study was devised innovatively to explore the role of DLL3 in the progression of SCLC. Immunohistochemistry (IHC) analysis was used to examine DLL3 expression in paraffin-embedded SCLC tumor samples. Upregulation of DLL3 reduced chemotherapy sensitivity. Kaplan-Meier analysis was used to analyze the progression-free survival or overall survival of SCLC patients with high or low level of DLL3. The negative association between DLL3 expression and the PFS or OS rate of SCLC patients was identified. Relative high level of DLL3 was determined in SCLC cell lines by using qRT-PCR analysis. Loss-of function assays were performed to detect the biological functions of the silencing of DLL3 in SCLC. As a result, silencing of DLL3 led to the proliferative and migratory inhibition of SCLC cells and reversed EMT process. Mechanistically, DLL3 mRNA was stabilized by the RNA-binding protein lin-28 homolog B (LIN28B). Further mechanism investigation revealed that LIN28B and DLL3 are two downstream targets of miR-518d-5p. Finally, rescue assays demonstrated that LIN28B and miR-518d-5p could regulate DLL3-mediated cell proliferation and migration. Collectively, our present study revealed a novel molecular pathway in SCLC, which providing a new insight in exploring the therapeutic strategy for SCLC.

The microRNAs let-7 and miR-9 down-regulate the axon-guidance genes Ntn1 and Dcc during peripheral nerve regeneration

Axon guidance helps growing neural axons to follow precise paths to reach their target locations. It is a critical step for both the formation and regeneration of neuronal circuitry. Netrin-1 (Ntn1) and its receptor, deleted in colorectal carcinoma (Dcc) are essential factors for axon guidance, but their regulation in this process is incompletely understood. In this study, using quantitative real-time RT-PCR (qRT-PCR) and biochemical and reporter gene assays, we found that the Ntn1 and Dcc genes are both robustly up-regulated in the sciatic nerve stump after peripheral nerve injury. Moreover, we found that the microRNA (miR) let-7 directly targets the Ntn1 transcript by binding to its 3'-untranslated region (3'-UTR), represses Ntn1 expression, and reduces the secretion of Ntn1 protein in Schwann cells. We also identified miR-9 as the regulatory miRNA that directly targets Dcc and found that miR-9 down-regulates Dcc expression and suppresses the migration ability of Schwann cells by regulating Dcc abundance. Functional examination in dorsal root ganglion neurons disclosed that let-7 and miR-9 decrease the protein levels of Ntn1 and Dcc in these neurons, respectively, and reduce axon outgrowth. Moreover, we identified a potential regulatory network comprising let-7, miR-9, Ntn1, Dcc, and related molecules, including the RNA-binding protein Lin-28 homolog A (Lin28), SRC proto-oncogene nonreceptor tyrosine kinase (Src), and the transcription factor NF-κB. In summary, our findings reveal that the miRs let-7 and miR-9 are involved in regulating neuron pathfinding and extend our understanding of the regulatory pathways active during peripheral nerve regeneration.

Trans-splicing of the C. elegans let-7 primary transcript developmentally regulates let-7 microRNA biogenesis and let-7 family microRNA activity.

The sequence and roles in developmental progression of the microRNA let-7 are conserved. In general, transcription of the let-7 primary transcript (pri-let-7) occurs early in development, whereas processing of the mature let-7 microRNA arises during cellular differentiation. In Caenorhabditiselegans and other animals, the RNA-binding protein LIN-28 post-transcriptionally inhibits let-7 biogenesis at early developmental stages, but the mechanisms by which LIN-28 does this are not fully understood. Nor is it understood how the developmental regulation of let-7 might influence the expression or activities of other microRNAs of the same seed family. Here, we show that pri-let-7 is trans-spliced to the SL1 splice leader downstream of the let-7 precursor stem-loop, which produces a short polyadenylated downstream mRNA, and that this trans-splicing event negatively impacts the biogenesis of mature let-7 microRNA in cis Moreover, this trans-spliced mRNA contains sequences that are complementary to multiple members of the let-7 seed family (let-7fam) and negatively regulates let-7fam function in trans Thus, this study provides evidence for a mechanism by which splicing of a microRNA primary transcript can negatively regulate said microRNA in cis as well as other microRNAs in trans.