Whole genomic size is about 70 kbp, but consists of 27 exons.

KNL1 mRNA expression is dominant in normal human testis and slight expression are observed in other organs, such as placenta. Analysis on cancer cell lines, such as HeLa, gave single band with 8.5 kb. There is another alternative splicing site at the 5 side of this gene that generates a short exon with 78 bp in cDNA. There are potential other alternative splicing at cancer cell lines.
Analysis on testis mRNA shows two bands with size of approximately 6 and 8,5 kb which are probably derived from the two isoforms.

Encodes 1833 amino acids and 2342 amino acids.
The KLN1 protein contains: conserved motifs, which are the following: a (S/G)ILK motif (aa 25-28), a RRVSF motif (aa 57-61), and, for BUB3 recognition, MELT repeats (aa 140-161, 308-329, 474-494, 562-582, 750-769, 859-882, 902-924, 940-962, 1025-1044, 1073-1096, 1114-1136, 1152-1174). The Bubs recognition KI motifs KI(D/N)XXXF(L/I)XXLK, are KIDTTSFLANLK (aa 202-213) for BUB1, and KIDFNDFIKRLK (aa 238-249) for BUB1B: (BUBR1); a nuclear localization signal (aa 1789-1803); a coiled coil region (aa 1942-2133) and the ZWINT (Zwint-1) binding region (aa 1834 or 19811 for a smaller region -2108); and RWD repeats (aa 2109- 2353) With the NSL1 (hMis14)-binding region (aa 2109-2316), according to Kiyomitsu et al., 2011, VEGA checking, and SwissProt.

KNL1 protein expressions with molecular weight of approximately 250 kDa and 300 kDa are observed in human testicular germ cells and cancer cell lines.

In germ cell of testis, significant high expressions of KNL1 protein are observed in nucleus of spermatocytes and slightly in spermatogonia. It is noteworthy that round spermatids express significantly high KNL1 protein in their pre-acrosome. As KNL1 protein has no hydrophobic signal peptide in its amino terminus, it probably localizes outer surface of pre-acrosome membrane of spermatids inside of the cells..
In mitosis KNL1 protein is localized in kinetochore in a human cancer cell line.

KNL1 is a large of kinetochore protein, constituting KMN (KNL1/ MIS12 complex/ NDC80 complex) network. KMN network is the central hub of outer kinetochore, not only connecting mitotic chromosomes and spindles but also coordinating microtubule-binding, chromosome congression and spindle assembly checkpoint (SAC) signaling. There are two microtubule binding activity in KMN network, one in NDC80 and the other in KNL1. KNL1 protein binds directly microtubule through its far N-terminal region, consisting of 68 amino acids, in vitro, and indirectly through the interactions with MIS12 and NDC80 complexes at its C-terminus. KNL1 depletion affects metaphase chromosome congression. In C. elegans,
The depletion preclude metaphase chromosomal congression, while in yeast, Drosophila and human cells, the depletions show partial alignment phenotypes, in which some chromosomes congress to the equator but many chromosomes remain stranded near the spindle poles. In the N-terminus of KNL1 protein, AURKB (Aurora kinase B) phosphorylates SILK and RVSF motifs, and then disrupts the interaction between KNL1 and protein phosphatase I (PP1), which also binds the motifs. KNL1 augments Aurora B kinase activity that phosphorylates outer kinetochore protein, such as NDC80, resulting in reduction its microtubule-binding activity. Protein phosphatase 2A ( PP2A) is recruited by BUB1B (BubR1), one of SAC protein. Both PP1 and PPA2 are suggested to counteract Aurora B kinase activity. Especially, PP1 was shown to stabilize microtubule attachments to kinetochores probably through KNL1, while Aurora B kinase destabilizes microtubule-binding by NDC80 phosphorylation as described. The destabilized binding of KNL1 to microtubules is presumably important for correcting and eliminating erroneous kinetochore-microtubule attachment during SAC.
It is suggested that the bindings with microtubule and with PP1 also play roles in SAC silencing. Although their binding sites are in close proximity, one of their bindings do not affect the other, and they contribute independently to the silencing of SAC. Two KI motifs, KI1 and KI2, localized in the N-terminus, bind SAC protein, BUB1 and BUB1B, respectively, through tetratricopeptide repeats (TPRs) of the proteins, resulting into folding the motifs into short alpha-helices. Although BUB1 fragment with mutation in KI-binding domain was not able to bind to KNL1 in vitro, BUB1 and BUB1B mutants with KI-binding sites were able to attach to kinetochore. Mutations in the BUB3-binding domain (BUB3-BD) in BUB1 and BUB1B prevent kinetochore localization of the SAC protein. BUB1 fragment consisting only of N-terminus with TPRs does not localize to kinetochore and longer fragments that accommodate BUB3-BD did. Those results suggest that BUB3-BD of BUB1 and BUB1B, rather than TPRs, is critical for their recruitment to kinetochore and that the interaction between TPRs of Bub proteins and KI-motif of KNL1 might play a subsidiary role in the localization of BUB1 and BUB1B to kinetochore. In response to SAC signal, the first step of this response is phosphorylation of MELT motifs of KNL1, located in the N-terminal and central region, by Mps1 kinase, and then the phosphorylated MELT motifs bind BUB3/BUB1complex, mediating SAC signaling. KNL1- BUB3-BUB1(KBB) complex binds MXD1 (MAD1)/ MAD2L1 (MAD2) complex, and then, together with MAD1 phosphorylation by Msp1, the binding leads to CDC20/MAD2 formation, an essential part of Mitotic Checkpoint Complex (MCC) that inhibits Anaphase Promoting Complex/Cyclosome (APC/C).
Recently, it suggested that there are two pathways for recruiting MAD1-MAD2 that results in SAC activation. One is the pathway through KBB, as described above, the other is KNTC1 (ROD)-RW10- ZWILCH, (RZZ) complex, which interacts with KNL1 through ZWINT (Zwint-1 protein). The former is required for SAC activation when kinetochores are misaligned but is not essential when kinetochores are unattached from microtubules. The latter binds SPDL1 (Spindly protein) and MAD1-MAD2, and causes the anaphase-onset delay in response to unattached kinetochore independently of the former.
It was suggested that the binding of KNL1 with microtubules and with PP1 contribute to silencing of SAC, in which motor protein dynein, moving along on microtubules, is suggested to work to strip MAD1-MAD2. The C-terminal region of KNL1 interacts with MIS12 and Zwint-1 protein, through RWD motif and coiled-coiled region, respectively. The binding with the former plays role in connecting inner kinetochore with KNL1 and the latter mediates the interaction of KNL1 with RZZ complex which works in SAC regulation as described above.