**5. Suppression of** *let-7* **miRNAs by LIN28**

LIN28 is a highly conserved RNA binding protein with two paralogues, LIN28A and LIN28B. Both LIN28A and LIN28B have a cold-shock domain (CSD) at the N-terminal and two zinc knuckle domains (ZKDs) at the C-terminal [113]. LIN28 promotes cell proliferation and inhibits cell differentiation [114]. LIN28 is also involved in reprogramming of differentiated somatic cells into tumor or stem cells, hence known as oncoprotein [115,116]. Reduced expression of LIN28 in embryos results in reduced prenatal growth and development and long-term metabolic abnormalities [117]. Knockout of LIN28A in mice leads to perinatal lethality while LIN28B knockout results in postnatal growth abnormalities in males. Knockout of both LIN28A and LIN28B in mice is embryonically lethal at around E13. Conditional knockout of LIN28A and LIN28B in mice at six weeks of age does not produce any evident phenotype [118]. Collectively, these findings show that LIN28 has a more profound role during prenatal development and organogenesis.

LIN28 regulates expression of several genes either directly binding to the mRNA of target genes or by repressing the production of mature *let-7* miRNAs, later being a more prevalent mechanism [118,119]. There are conflicting theories about the localization of LIN28A and LIN28B in cells. LIN28A is predominantly localized in the cytoplasm but can be found in the nucleus as well [120,121]; however, according to another study, LIN28A is exclusively localized in the cytoplasm [122]. LIN28B has a nucleolar and nuclear localization signal, while others found it predominantly in cytoplasm with a possibility to shuttle to the nucleus [113,119,123]. LIN28A and LIN28B selectively repress the expression and maturation of *let-7* miRNAs by distinct mechanisms, without directly affecting the expression of other miRNAs [124]. LIN28A CSD binds GNGAY motif while ZKDs bind GGAG motif in the stem loop pre-*let-7* miRNA in the cytoplasm. After binding to pre-*let-7* miRNA, LIN28A recruits terminal uridylyl transferase (TUTase) Zcchc11 (also referred as TUT4). TUT4 causes polyuridylation of pre-*let-7* miRNA which blocks the cleavage of pre-*let-7* miRNA by Dicer and hence inhibits the production of mature *let-7* miRNAs [124–126]. Polyuridylated pre-*let-7* miRNA is recognized and degraded by exonuclease Dis3L2 [127]. The mechanism of *let-7* miRNA suppression by LIN28B remains controversial and there are four different theories. First, LIN28B inhibits maturation of *let-7* miRNAs by TUT4 independent mechanism. In the nucleus, LIN28B binds the pri-*let-7* miRNA by its CSD and ZKDs and inhibits its processing by a microprocessor [122]. Second, in the cytoplasm, LIN28B binds to pre-*let-7* miRNA and inhibits its processing by Dicer [128]. Third, in the cytoplasm, LIN28B binds to pre-*let-7* miRNA and leads to its polyuridylation by recruiting an unknown TUTase, leading to its degradation [121]. Fourth, in the nucleolus, LIN28B has the ability to sequester pri-*let-7* miRNAs and hence inhibits further processing to mature pre-*let-7* miRNAs [122].

In 2018, Ustianenko et al. demonstrated that LIN28 selectively regulates a subclass of *let-7* miRNAs [129]. Using single nucleotide resolution, they identified –(U)GAU- as the new binding motif of the CSD. Some pre-*let-7* miRNAs with both (U)GAU and GGAG motifs in the stem loop make a stronger and stable interaction with LIN28 and are referred to as CSD+, while the others which do not contain (U)GAU motif are called CSD- . The CSD<sup>+</sup> subclass includes pre-*let-7b,* pre-*let-7d,* pre-*let-7f-1,* pre-*let-7g,* pre-*let-7i*, and *miR-98.* The CSDsubclass includes pre-*let-7a-1,* pre-*let-7a-2,* pre-*let-7-3,* pre-*let-7c,* pre-*let-7e*, and pre-*let-7f-2* [110,129]. Although all *let-7* miRNAs express ZKD binding GGAG motif, both LIN28A and LIN28B have shown greater binding affinity for CSD<sup>+</sup> pre-*let-7* miRNAs, hence leading to their polyuridylation and repression [129].
